bluuebunny/crossref_metadata_2025 huggingface dataset:
Dermatitis as a component of the fetal inflammatory response syndrome is associated with activation of Toll‐like receptors in epidermal keratinocytes
Authors: Kim Y M , Romero R , Chaiworapongsa T , Espinoza J , Mor G , Kim C J
Published: October 2006

Aims Microbial invasion of the amniotic cavity (MIAC) elicits a fetal inflammatory response such as funisitis and chorionic vasculitis. However, little is known about the changes of fetal skin during MIAC. Toll‐like receptors recognize microbial products and initiate an immune response. The aims of this study were to examine histopathological features of fetal skin exposed to MIAC and to assess the changes in Toll‐like receptor (TLR)‐2 and TLR‐4 expression.

Methods and results Skin samples were obtained from fetal autopsies (n = 12). The cases were classified according to the presence (n = 8) or absence (n = 4) of acute chorioamnionitis and analysed by immunohistochemistry using a panel of antibodies. Leucocytic infiltrates into the superficial dermis were observed in cases with chorioamnionitis; the majority of inflammatory cells were neutrophils, lymphocytes and histiocytes. TLR‐2 immunoreactivity in the skin was stronger in fetuses with chorioamnionitis than in those without this condition. However, immunoreactivity of TLR‐4 in the fetal skin was constitutively expressed, regardless of the presence or absence of chorioamnionitis.

Conclusions This study demonstrates for the first time that fetal dermatitis can be detected and is part of the fetal inflammatory response syndrome (FIRS). We propose that this ‘FIRS‐associated fetal dermatitis’ is a fetal counterpart of chorioamnionitis.
Regulatory noise in prokaryotic promoters: how bacteria learn to respond to novel environmental signals
Authors: de Lorenzo Victor , Pérez‐Martín José
Published: October 2006

Various features of the regulation of pathways for biodegradation of recalcitrant compounds by Pseudomonas provide insights into the mechanisms by which operons evolve to acquire conditionally active promoters that permit the corresponding genes to be transcribed only when required. The regulatory noise hypothesis’proposes that transcriptional control systems develop responsiveness to new signals due to the leakiness and lack of specificity of preexisting promoters and regulators. When needed, these may become more specific through suppression of undesirable signals and further fine‐tuning of the recruited proteins to interact with distinct chemicals. This hypothesis is supported by the sophisticated regulation of α54‐dependent promoters of the TOL (toluene biodegradation) operons, which can be activated to various degrees by heterologous proteins. Such illegitimate’activation is suppressed by bent DNA structures, either static or protein induced, between promoter core elements. Therefore, not only the regulators but also the DNA sequences participate in the process that gives rise to novel specificities.
Extracellular addition of a domain of HIV‐1 Vpr containing the amino acid sequence motif H(S/F)RIG causes cell membrane permeabilization and death
Authors: Macreadie Ian G. , Arunagiri Chinniah K. , Hewish Dean R. , White Jacinta F. , Azad A. A.
Published: October 2006

Vpr is a virion‐associated protein of human immuno‐deficiency virus type 1 (HIV‐1) whose function in acquired immune deficiency syndrome (AIDS) has been uncertain. We previously employed yeast as a model to examine the effects of Vpr on basic cellular functions; intracellular Vpr was shown to cause cell‐growth arrest and structural defects, and these effects were caused by a region of Vpr containing the sequence HFRIGCRHSRIG. Here we show that peptides containing the H(S/F)RIG amino acid sequence motif cause death when added externally to a variety of yeast including Saccharomyces cerevisiae, Kluyveromyces lactis, Candida glabrata, Candida albicans and Schizosaccharomyces pombe. Such peptides rapldly entered the cell from the time of addition, resulting in cell death. Elevated levels of ions, particularly magnesium and calcium ions, abrogated the cytotoxic effect by preventing the Vpr peptides from entering the cells. Extracellular Vpr found in the serum, or breakdown products of extracellular Vpr, may have similar effects to the Vpr peptides described here and could explain the death of uninfected by‐stander cells during AIDS.
Saccharomyces cerevisiae TEC1 is required for pseudohyphal growth
Authors: Gavrias Victoria , Andrianopoulos Alex , Gimeno Carlos J. , Timberlake William E.
Published: October 2006

Diverse eukaryotic organisms share developmental transcription factors with homologous DNA‐binding domains. We showed that the developmental regulator AbaA, a member of the ATTS/TEA (AbaA, TEF‐1, TEC1, Scalloped/TEF‐1, TEC1, AbaA) class of transcription factors of the filamentous fungus Aspergillus nidulans, induces pseudohyphal development in the yeast Saccharomyces cerevisiae. The S. cerevisiae homologue of AbaA, TEC1p, is required for this morphological transition. We provide evidence that TEC1p functions in co‐operation with STE12p to induce pseudohyphal development.
Characterization of the distal promoter element of halobacteria in vivo using saturation mutagenesis and selection
Authors: Danner Stefan , Soppa Jörg
Published: October 2006

The sequence and spacing requirements of the archaeal‘distal promoter element’(DPE) were examined by randomizing positions ‐19 to ‐32 upstream of the transcriptional start site of the ferredoxin (fdx) promoter of Halobacterium salinarium. This randomized promoter library containing 414 entries was cloned in front of the dihydrofolate reductase (DHFR) reporter gene and transformed into Haloferax volcanii. Two approaches were used to characterize these synthetic promoters. First, 1040 independent clones were randomly chosen and their degrees of trimethoprim resistance were determined. The sequences of 20 clones that were either sensitive, partially resistant or very resistant, respectively, were determined. Secondly, the transformed library was screened by direct selection for high‐activity promoters by growing transformants in the presence of trimethoprim. Both approaches produced the following consensus sequence for a halobacterial promoter: ‐32 RGTWWWWRACYGSY ‐19 (where R = A or G; Y = C or T; W = A or T; S = G or C; N = A, C, G or T). Further characterization of two sensitive, two partially resistant, and two very resistant clones verified that DHFR activity and cell phenotype are directly correlated. Sensitive clones did not contain detectable dhfr mRNA, whereas partially resistant clones contained a 700 nucleotide (nt)‐long transcript, and very resistant clones contained both the 700 nt‐long transcript and a second, more abundant, 500 nt‐long truncated transcript. Quantification of the dhfr mRNA and DHFR enzyme activity suggests that the 3’‐untranslated region of the dhfr transcript, missing from the shorter transcript, functions as a negative regulator of translation.
Bacterial signalling involving eukaryotic‐type protein kinases
Authors: Zhang Cheng‐Cai
Published: October 2006

Protein Ser, Thr and Tyr kinases play essential roles in signal transduction in organisms ranging from yeast to mammals, where they regulate a variety of cellular activities. During the last few years, a number of genes that encode eukaryotic‐type protein kinases have also been identified in four different bacterial species, suggesting that such enzymes are also widespread in prokaryotes. Although many of them have yet to be fully characterized, several studies indicate that eukaryotic‐type protein kinases play important roles in regulating cellular activities of these bacteria, such as cell differentiation, pathogenicity and secondary metabolism. A model based on the possible coupling between two‐component systems and eukaryotic‐type protein kinases is proposed to explain the function of eukaryotic‐type protein kinases in bacterial signalling in the light of studies in bacteria, as well as in plants and yeast. These two groups of eukaryotes possess signal‐transduction pathways involving both two‐component systems and eukaryotic protein kinases.
Molecular biology of iron acquisition in Saccharomyces cerevisiae
Authors: Askwith Candice C. , de Silva Deepika , Kaplan Jerry
Published: October 2006

In recent years, significant advances have been made in our understanding of the mechanism and regulation of elemental iron transport in the eukaryote Saccharomyces cerevisiae. This organism employs two distinct iron‐transport systems, depending on the bioavailability of the metal. In iron‐replete environments, a low‐affinity transport system (Km=30μM) is used to acquire iron. This system may also be used to acquire other metals including cobalt and cadmium. When environmental iron is limiting, a high‐affinity (Km=0.15 μM) iron‐transport system is induced. Genetic studies in S. cerevisiae have identified multiple genes involved in both iron‐transport systems. Cell‐surface reductases, FRE1 and FRE2, provide ferrous iron for both systems. A non‐ATP‐dependent transmembrane transporter (FET4) has been identified as the main component of low‐affinity transport. One gene identified to date as part of the high‐affinity transport system is FET3, which shows high sequence and functional homology to multicopper oxidases. Accessory genes required for the functioning of this transport system include a plasma‐membrane copper transporter (CTR1), an intracellular copper transporter (CCC2), and a putative transcription factor (AFT1). The mechanism by which these genes act in concert to ensure iron accumulation in S. cerevisiae presents an intriguing picture, drawing parallels with observations made in the human system almost 40 years ago.
The sxa2‐dependent inactivation of the P‐factor mating pheromone in the fission yeast Schizosaccharomyces pombe
Authors: Ladds Graham , Rasmussen Erik Michael , Young Tom , Nielsen Olaf , Davey John
Published: October 2006

Haploid cells of the fission yeast Schizosaccharomyces pombe exist in one of two mating types, referred to as M and P. Conjugation occurs between cells of opposite mating type and is controlled by the reciprocal action of diffusible pheromones. Loss of function of the sxa2 gene in M cells causes hypersensitivity to the P‐factor mating pheromone and a reduction in mating efficiency. Here we demonstrate the secretion of an sxa2‐dependent carboxypeptidase that inactivates P‐factor by removal of the C‐terminal leucine residue.
The bundle‐forming pili of enteropathogenic Escherichia coli: transcriptional regulation by environmental signals
Authors: Puente Jose Luis , Bieber David , Ramer Sandra W. , Murray William , Schoolnik Gary K.
Published: October 2006

The bundle‐forming pili (BFP) of enteropathogenic Escherichia coli (EPEC) are required for the development of circumscribed colonies of bacteria attached to the surfaces of cultured epithelial cells, a process termed the localized adherence (LA) phenotype. Similar lesions are evident in jejunal biopsies from EPEC‐infected children. BFP production is not constitutive, but instead occurs upon transfer of bacteria from nutrient broth to tissue culture media, indicating that the expression of BFP may be environmentally regulated. To learn more about how BFP protein expression is induced during epithelial‐cell adherence, bfpA‐cat transcriptional fusions and northern blot analyses were employed to monitor bfpA expression as a function of environmental signals and growth kinetics. bfpA expression was found to be regulated at the transcriptional level, and to require a separate locus on the EPEC adherence factor (EAF) plasmid. Expression occurred selectively during exponential‐growth phase and was greatest between 35 and 37°C, and in the presence of calcium. Ammonium (20 mM) significantly reduced bfpA mRNA and protein expression and the development of the LA phenotype. Analysis of the bfpA upstream sequence and identification of the transcription initiation site revealed a conventional σ70‐dependent promoter and an AT‐rich tract that might affect promoter activity. Taken together, these findings further support the pathogenic role of BFP
Involvement of N‐acyl‐l‐homoserine lactone autoinducers in controlling the multicellular behaviour of Serratia liquefaciens
Authors: Eberl Leo , Winson Michael K. , Sternberg Claus , Stewart Gordon S. A. B. , Christiansen Gunna , Chhabra Siri Ram , Bycroft Barrie , Williams Paul , Molin Søren , Givskov Michael
Published: October 2006

Several bacterial species possess the ability to differentiate into highly motile swarmer cells capable of rapid surface colonization. In Serratia liquefaciens, we demonstrate that initiation of swarmer‐cell differentiation involves diffusible signal molecules that are released into the growth medium. Using high‐performance liquid chromatography (HPLC), high resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, we identified N‐butanoyl‐l‐homoserine lactone (BHL) and N‐hexanoyl‐l‐homoserine lactone (HHL) in cell‐free Serratia culture supernatants. BHL and HHL are present in a ratio of approximately 10:1 and their structures were unequivocally confirmed by chemical synthesis. The swrlswarmer initiation) gene, the predicted translation product of which exhibits substantial homology to the Luxl family of putative Nacyl homoserine lactone (AHL) synthases is responsible for directing synthesis of both BHL and HHL. In an swrl mutant, swarming motility is abolished but can be restored by the addition of an exogenous AHL. These results add swarming motility to the rapidly expanding list of phenotypes known to be controlled through quorum sensing.
Conserved amino acids in the N‐ and C‐terminal domains of integral membrane transporter FhuB define sites important for intra‐ and intermolecular interactions
Authors: Böhm Brigitte , Boschert Hartmut , Köster Wolfgang
Published: October 2006

Transport of iron(III) hydroxamates across the inner membrane of Escherichia coli is mediated by a periplasmic binding protein‐dependent transport (PBT) mechanism. FhuB, the integral membrane component of the system, is composed of covalently linked halves (FhuB[N] and FhuB[C]) which still function when present as two distinct polypeptide chains. Our analysis of two uptake‐deficient FhuB derivatives provides evidence for a mechanistically novel type of functional complementation:‘domain displacement’ in the cytoplasmic membrane. Amino acid residues 60 and 426 in the FhuB polypeptide chain may define key positions that are important for FhuB[N]–FhuB[C] interaction. Furthermore, FhuB derivatives, altered in either one of their conserved regions ‐ typical of PBT related integral membrane proteins ‐ displayed a dominant negative effect on ferric hydroxamate transport. The experimental data suggest that the two functionally equivalent conserved regions in FhuB[N] and FhuB[C] are primarily involved in the interaction with another component of the transport system, probably FhuC.
Genetic organization and functional analysis of the otn DNA essential for cell‐wall polysaccharide synthesis in Vibrio cholerae O139
Authors: Bik Elisabeth M. , Bunschoten Annelies E. , Willems Rob J. L. , Chang Annie C. Y. , Mooi Frits R.
Published: October 2006

In 1992 a new Vibrio cholerae strain, designated V. cholerae O139 Bengal, emerged which has been responsible for large outbreaks of cholera in India and Bangladesh. Previously, we have shown that this strain arose from a V. cholerae O1 strain by the acquisition of novel DNA. Sequence analysis revealed that the novel DNA is flanked by two genes, rfaD and rfbQRS, which are also found in O1 strains. The mosaic structure of rfaDvco139 indicated that it was one of the regions involved in recombination between donor and acceptor DNA. However, sequence divergence between the O1 and O139 rfbQRS genes indicated that the second recombination site between donor and O1‐acceptor DNA is probably located downstream of rfbDvco139. The DNA region between rfaDvco139 and rfbQRSvco139, designated otn, contained seven open reading frames (ORFs). Two ORFs, otnA and otnB, showed homology with genes involved in cell‐wall polysaccharide synthesis. Mutations in otnA and otnB indicated that they are required for capsule synthesis but not lipopolysaccharide synthesis. The otn DNA is also found inV. cholerae O69 and O141 strains, and the organization of this DNA was essentially identical to that in the O139 strain. However, sequence divergence of the otnAB genes indicated that the O139 otn DNA region was not derived from the O69 or O141 strains. No antigenic relationship was found between the different V. cholerae serotypes carrying otn DNA, so the genes determining the antigenic specificity of the O antigen or capsule must be located outside the otn DNA. The O139 otn DNA contained a JUMPstart sequence, which is associated with polysaccharide‐synthetic genes in several bacterial species. Furthermore, a repeat motif was observed in extragenic regions. A number of observations suggest that these sequences may facilitate gene flow between V. cholerae strains and the assembly of clusters of functionally related genes.
Protein engineering studies of A‐chain loop 47‐56 of Escherichia coli heat‐labile enterotoxin point to a prominent role of this loop for cytotoxicity
Authors: Feil Ingeborg K. , Reddy Raghava , de Haan Lolke , Merritt Ethan A. , van den Akker Focco , Storm Daniel R. , Hol Wim G. J.
Published: October 2006

Heat‐labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240‐residue A chain and five 103‐residue B subunits. The B pentamer is responsible for GM1 receptor recognition, whereas the A subunit carries out an ADP‐ribosylation of an arginine residue in the G protein, GSα, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47–56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild‐type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single‐site substitutions have been made in the LT‐A subunit 47–56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild‐type residues Thr‐50 and Val‐53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active‐site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine‐substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild‐type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild‐type LT. These results lend support for assignment of a prominent role to loop 47–56 in catalysis by LT and CT.
Nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of the gene and its role in cellular growth and exopolysaccharide alginate synthesis
Authors: Sundin George W. , Shankar Sandeep , Chugani Sudha A. , Chopade B. A. , Kavanaugh‐Black Andrew , Chakrabarty A. M.
Published: October 2006

We report the cloning and determination of the nucleotide sequence of the gene encoding nucleoside diphosphate kinase (Ndk) from Pseudomonas aeruginosa. The amino acid sequence of Ndk was highly homologous with other known bacterial and eukaryotic Ndks (39.9 to 58.3% amino acid identity). We have previously reported that P. aeruginosa strains with mutations in the genes algR2 and algR2 algH produce extremely low levels of Ndk and, as a consequence, are defective in their ability to grow in the presence of Tween 20, a detergent that inhibits a kinase which can substitute for Ndk. Hyperexpression of ndk from the clone pGWS95 in trans in the P. aeruginosa algR2an6 algR2 algH double mutant restored Ndk production to levels which equalled or exceeded wild‐type levels and enabled these strains to grow in the presence of Tween 20. Hyperexpression of ndk from pGWS95 in the P. aeruginosa algR2 mutant also restored alginate production to levels that were approximately 60% of wild type. Nucleoside diphosphate kinase activity was present in both the cytosolic and membrane‐associated fractions of P. aeruginosa. The cytosolic Ndk was non‐specific in its transfer activity of the terminal phosphate from ATP to other nucleoside diphosphates. However, the membrane form of Ndk was more active in the transfer of the terminal phosphate from ATP to GDP resulting in the predominant formation of GTP. We report in this work that pyruvate kinase and Ndk form a complex which alters the specificity of Ndk substantially to GTP. The significance of GTP in signal transduction
Evidence for trehalose‐6‐phosphate‐dependent and ‐independent mechanisms in the control of sugar influx into yeast glycolysis
Authors: Hohmann Stefan , Bell Walter , NevesA Maria Jose , Valckx Dirk , Thevelein Johan M.
Published: October 2006

In the yeast Saccharomyces cerevisíae, trehalose‐6‐phosphate (tre‐6‐P) synthase encoded by GGS1/TPS1, is not only involved in the production of trehalose but also in restriction of sugar influx into glycolysis in an unknown fashion; it is therefore essential for growth on glucose or fructose. In this work, we have deleted the TPS2 gene encoding tre‐6‐P phosphatase in a strain which displays very low levels of Ggs1/Tps1, as a result of the presence of the byp1‐3 allele of GGS1/TPS1. The byp1‐3 tps2Δ double mutant showed elevated tre‐6‐P levels along with improved growth and ethanol production, although the estimated concentrations of glycolytic metabolites indicated excessive sugar influx. In the wild‐type strain, the addition of glucose caused a rapid transient increase of tre‐6‐P. In tps2Δ mutant cells, which showed a high tre‐6‐P level before glucose addition, sugar influx into glycolysis appeared to be diminished. Furthermore, we have confirmed that tre‐6‐P inhibits the hexokinases in vitro. These data are consistent with restriction of sugar influx into glycolysis through inhibition of the hexokinases by tre‐6‐P during the switch to fermentative metabolism. During logarithmic growth on glucose the tre‐6‐P level in wild‐type cells was lower than that of the byp1‐3 tps2Δ. mutant. However, the latter strain arrested growth and ethanol production on glucose after about four generations. Hence, other mechanisms, which also depend on Ggs1/Tps1, appear to control sugar influx during growth on glucose. In addition, we provide evidence that the requirement for Ggs1/Tps1 for sporulation may be unrelated to its involvement in trehalose metabolism or in the system controlling glycolysis.
The nd7‐binding protein (Nbp) regulates the ndh gene of Escherichia coti in response to growth phase and is identical to Fis
Authors: Green J. , Anjum M. F. , Guest J. R.
Published: October 2006

The ndh gene that encodes the non‐proton‐translocating NADH dehydrogenase II of Escherichia coli is anaerobically repressed by FNR. However, in the absence of FNR, ndh expression is enhanced by anaerobic growth in media containing amino acids. Two potential regulatory proteins that may be associated with this activation have previously been detected, Arr (amino acid response regulator) and Nbp (ndh‐bind'mg protein). Studies with the heat‐stable Nbp have now shown that it is present in E. coli grown both aerobicaliy and anaerobically in rich and minimal media, indicating that it is not specifically associated with the anaerobic enhancement of ndh expression. The Nbp activity of aerobic cultures was maximal during exponential growth phase (when ndh promoter activity is minimal) but fell rapidly as cultures entered stationary phase and ndh expression increased. Protein purification and mutant studies have further shown that Nbp is identical to the Fis protein (factor for inversion stimulation). Three major and two minor Nbp (Fis)‐binding sites have been identified in the ndh promoter by gel retardation and DNase I footprinting. The major sites are centred at ‐123, 72 and +51, in decreasing order of binding affinity. At low concentrations, Nbp (Fis) increased transcription from the ndh promoter by up to 25%, whereas at higher concentrations it prevented RNA polymerase (RNAP) binding and open complex formation. Consequently, Nbp (Fis) can both activate and repress transcription from the ndh promoter. The results suggest that Nbp (Fis) serves to ensure that the energetically efficient proton‐translocating NADH dehydrogenase I is used in preference to the non‐proton translocating NADH dehydrogenase II during periods of rapid growth, by repressing expression of the ndh gene.
Molecular basis of intercellular adhesion in the biofilm‐forming Staphylococcus epidermidis
Authors: Heilmann Christine , Schweitzer Oliver , Gerke Christiane , Vanittanakom Nongnuch , Mack Dietrich , Götz Friedrich
Published: October 2006

The Staphylococcus epidermidis genes icaABC are involved in the synthesis of the polysaccharide intercellular adhesin (PIA), which is located mainly on the cell surface, as shown by immunofluorescence studies with PIA‐specific antiserum. PIA was shown to be a linear β‐1,6‐linked glucosaminoglycan composed of at least 130 2‐deoxy‐2‐amino‐D‐glucopyrano‐syl residues of which 80–85% are N‐acetylated, the rest being non‐N‐acetylated and positively charged. A transposon insertion in the icaABC gene cluster (ica, intercellular adhesion) led to the loss of several traits, such as the ability to form a biofilm on a polystyrene surface, cell aggregation, and PIA production. The mutant could be complemented by transformation with the IcaABC‐carrying plasmid pCN27. Transfer of pCN27 into the heterologous host Staphylococcus carnosus led to the formation of large cell aggregates, the formation of a biofilm on a glass surface, and PIA expression. The nucleotide sequence of icaABC suggests that the three genes are organized in an operon and that they are co‐transcribed from the mapped ica A promoter. Ica A contains four potential transmembrane helices, indicative of a membrane location. The deduced Ica A sequence shows similarity to those of polysaccharide‐polymerizing enzymes, the most pronounced being with a Rhizobium meliloti N‐acetylglucosaminyltransferase involved in lipo‐chitin biosynthesis (22.5% overall identity and 37.4% overall similarity). This similarity suggests that Ica A has N‐acetylglucosaminyltransferase activity in the formation
Overlaps and parallels in the regulation of intrinsic multiple‐antibiotic resistance in Escherichia coli
Authors: Miller Paul F. , Sulavik Mark C.
Published: October 2006

Chromosomally encoded systems present in a variety of bacteria appear to play a central role in determining the intrinsic level of resistance to many commonly used antibiotics. Work with the Gram‐negative bacterium Escherichia coli has shown that there is significant similarity at the amino acid sequence level among the structural components of these resistance systems as well as among their genetic regulators. This review describes two of the better‐studied regulatory systems, marRAB and soxRS, as well as two regulated multidrug‐efflux systems, encoded by emrAB and acrAB, and focuses on conserved themes in their primary structures and environmental stimuli. The observed resistance to clinically important antibiotics appears to reflect an overlap with broad‐ranged adaptive responses by free‐living bacteria to noxious plant materials in their natural environment.
Intracellular endosymbiotic bacteria of Camponotus species (carpenter ants): systematics, evolution and ultrastructural characterization
Authors: Schröder Doris , Deppisch Heike , Obermayer Malu , Krohne Georg , Stackebrandt Erko , Hölldobler Bert , Goebel Werner , Gross Roy
Published: October 2006

Intracellular endosymbiotic bacteria inherent to ants of the genus Camponotus were characterized. The bacteria were localized in bacteriocytes, which are specialized cells of both workers and queen ants; these cells are intercalated between epithelial cells of the midgut. The bacteriocytes show a different morphology from the normal epithelial cells and carry a large number of the rod‐shaped Gram‐negative bacteria free in the cytoplasm. The bacteria were never observed in the neighbouring epithelial cells, but they were found intracellularly in oocytes, strongly indicating a maternal transmission of the bacteria. The 16S DNA encoding rrs loci of the endosymbionts of four species of the genus Camponotus derived either from Germany (C. herculeanus and C. ligniperdus), North America (C. floridanus) or South America (C. rufipes) were cloned after polymerase chain reaction (PCR) amplification using oligonucleotides complementary to all so far known eubacterial rrs sequences. The DNA sequences of the rrs loci of the four endosymbionts were determined, and, using various genus‐ and species‐specific oligonucleotides derived from variable regions in the rrs sequences, the identity of the bacteria present in the bacteriocytes and the ovarian cells was confirmed by PCR and in situ hybridization techniques. Comparison of the 16S DNA sequences with the available database showed the endosymbiotic bacteria to be members of the γ‐subclass of Proteobacteria. They formed a distinct taxonomic group, a sister taxon of the taxons defined by the tsetse fly and aphid endosymbionts. Within the γ‐subclass, the cluster of the ant, tsetse fly and aphid endosymbionts are placed adjacent to the family of Enterobacteriaceae. The evolutionary tree of the ant endosymbionts reflects the systematic classification and geographical distribution of their host insects, indicating an early co‐evolution of the symbiotic partners and a vertical transmission of the bacteria.
The positions of the sigma‐factor genes, whiG and sigF, in the hierarchy controlling the development of spore chains in the aerial hyphae of Streptomyces coelicolor A3(2)
Authors: Kelemen Gabriella H. , Brown Gary L. , Kormanec Ján , Potúčkova Laura , Chater Keith F. , Buttner Mark J.
Published: October 2006

whiG and sigF encode RNA polymerase sigma factors required for sporulation in the aerial hyphae of Streptomyces coelicolor. Their expression was analysed during colony development in wild‐type and sporulation‐defective whi mutant strains. Each gene was transcribed from a single promoter. Unexpectedly, whiG mRNA was present at all time points, including those taken prior to aerial mycelium formation; this suggests that whiG may be regulated post‐transcriptionally. Transcription of whiG did not depend upon any of the six known‘early’whi genes required for sporulation septum formation (whiA, B, G, H, /and J), placing it at the top of the hierarchy of whi loci. sigF expression appeared to be regulated at the level of transcription; sigF transcripts were detected transiently when sporulation septa were observed in the aerial hyphae. Transcription of sigF depended upon all six of the early whi genes, including whiG. The sigF promoter does not resemble the consensus sequence established for σWhiG‐dependent promoters and EnWhiG did not transcribe from the sigF promoter in vitro. Consequently, the genetic dependence of sigF upon whiG is very likely to be indirect. These results show that there is a hierarchical relationship between sigma factors required for Streptomyces sporulation and also that at least five other genes are involved in this transcriptional network.
Transcriptional activator of phage φ29 late promoter: mapping of residues involved in interaction with RNA polymerase and in DNA bending
Authors: Mencía Mario , Monsalve María , Salas Margarita , Rojo Fernando
Published: October 2006

Phage φ29 regulatory protein p4 activates transcription from the late A3 promoter by stabilizing σA‐RNA polymerase at the promoter as a closed complex. Activation requires interaction between both proteins. Protein p4 bends the DNA upon binding. We have performed a detailed mutagenesis study of the carboxyl end of the protein, which is involved in both transcription activation and DNA bending. The results indicate that Arg‐120 is the most critical residue for activation, probably mediating the interaction with RNA polymerase. Several basic residues have been identified, including Arg‐120, that contribute to maintenance of the DNA bending, probably via electrostatic interactions with the DNA backbone. The degree or stability of the induced bend apparently relies on the additive contribution of all basic residues of the carboxyl end of the protein. Therefore, the activation and DNA bending surfaces overlap, and Arg‐120 should interact with both DNA and RNA polymerase. As we show that protein p4 is a dimer in solution, and is bound to DNA as a tetramer, the results suggest a model in which two of the p4 subunits interact with the DNA, bending it, while the other two subunits remain accessible to interact with RNA polymerase.
Purification of recombinant Chlamydia trachomatis histone H1‐like protein Hc2, and comparative functional analysis of Hc2 and Hc1
Authors: Pedersen Lotte Bang , Birkelund Svend , Christiansen Gunna
Published: October 2006

The metabolically inactive developmental form of Chlamydia trachomatis, the elementary body, contains two very basic DNA‐binding proteins with homology to eukaryotic histone H1. One of these, Hc1, is relatively well characterized and induces DNA condensation in vitro, whereas the other, Hc2, is functionally virtually uncharacterized. In this study we describe the purification of Hc2, and a detailed comparative functional analysis of Hc2 and Hc1 is presented. By gel shift assays and electron microscopy, marked differences in the nucleic acid‐binding properties of Hc2 and Hc1 were observed. Furthermore, Hc2 was found to strongly inhibit translation and transcription in vitro. Our results imply that DNA condensation is not the only function of Hc2.
Tandem genes encode cell‐surface polypeptides SspA and SspB which mediate adhesion of the oral bacterium Streptococcus gordonii to human and bacterial receptors
Authors: Demuth Donald R. , Duan Yang , Brooks Wanda , Holmes Ann R. , McNab Roderick , Jenkinson Howard F.
Published: October 2006

The highly conserved antigen I/II family of polypeptides produced by oral streptococci are believed to be colonization determinants and may mediate adhesion of bacterial cells to salivary glycoproteins adsorbed to cells and tissues in the human oral cavity. Streptococcus gordonii is shown to express, on the cell surface, two antigen I/II polypeptides designated SspA and SspB (formerly Ssp‐5) that are the products of tandemly arranged chromosomal genes. The structure and arrangement of these genes is similar in two independently isolated strains, DL1 and M5, of S. gordonii. The mature polypeptide sequences of M5 SspA (1539 amino acid (aa) residues) and SspB (1462 aa residues) are almost wholly conserved (98% identical) in the C‐terminal regions (from residues 796 in SspA and 719 in SspB, to the respective C‐termini), well‐conserved (84%) at the N‐terminal regions (residues 1–429), and divergent (only 27% identical residues) within the intervening central regions. Insertional inactivation of the sspA gene in S. gordonii DL1 resulted in reduced binding of cells to salivary agglutinin glycoprotein (SAG), human erythrocytes, and to the oral bacterium Actinomyces naeslundii. Further reductions in streptococcal cell adhesion to SAG and to two strains of A. naeslundii were observed when both sspA and sspB genes were inactivated. The results suggest that both SspA and SspB polypeptides are involved in adhesion of S. gordonii cells to human and bacterial receptors.
Regulation by low temperatures and anaerobiosis of a yeast gene specifying a putative GPI‐anchored plasma membrane
Authors: Donzeau Mariel , Bourdineaud Jean‐Paul , Lauquin Guy J.‐M.
Published: October 2006

Expression of the yeast Saccharomyces cerevisiae SRP1 (serine‐rich protein) gene is shown here to be induced both‐ by low temperature and anaerobic growth conditions. We show that anaerobic SRP1 expression is haem‐dependent; however, haem influence does not operate through the action of the hypoxic‐gene ROX1 repressor. The SRP1 promoter region displaying the stress‐responsive elements is restricted to its first 551 bp, upstream of the initiation codon, although an upstream activation site contained in upstream sequences is required for full promoter activity. In addition, we demonstrate that the TIP1 gene, sharing similar nucleotide and polypeptide structure with SRP1, and previously reported to be a cold‐shock‐inducible gene, is also a hypoxic gene. Srp1 protein production is similarly induced by low temperature and anaerobic growth conditions. This protein, detected in the plasma membrane fraction, is shown to be exposed on the cell surface via a glycosyl‐phosphatidylinositol membrane anchoring.
Bacterial cytochromes P‐450
Authors: Munro Andrew W. , Lindsay J. Gordon
Published: October 2006

The cytochromes P‐450 (P‐450s) constitute an extremely large family ('superfamily') of haemoproteins that catalyse the oxidation of a wide range of physiological and non‐physiological compounds. A remarkable feature of the P‐450s is the manipulation of the same basic structure and chemistry to achieve an enormous range of functions in organisms as diverse as bacteria and man. Indeed, the P‐450s have been described as ‘the most versatile biological catalyst known’. Much research is focussed on mammalian P‐450s, with their roles in such processes as steroid transformations and the metabolism of carcinogens and other xenobiotics. However, our knowledge of the structure and function of the P‐450s has been advanced by analysis of a limited number of its bacterial members, primarily P‐450cam from Pseudomonas putida. Four P‐450 structures have been solved to date, all of which are from bacterial sources. The aim of this review is to assess current knowledge of the many bacterial P‐450s, with emphasis on their diverse biological roles and on the advances in our knowledge of this extremely important enzyme class, which have been made feasible through their study.
Structural and functional analysis of aa3‐type and cbb3‐type cytochrome c oxidases of Paracoccus denitrificans reveals significant differences in proton‐pump design
Authors: de Gier Jan‐Willem L. , Schepper Mike , Reijnders Willem N. M. , van Dyck Stef J. , Slotboom Dirk Jan , Warne Antony , Saraste Matti , Krab Klaas , Finel Moshe , Stouthamer Adriaan H. , van Spanning Rob J. M. , van der Oost John
Published: October 2006

In Paracoccusdenitrificans the aa3‐type cytochrome c oxidase and the bb3‐type quinol oxidase have previously been characterized in detail, both biochemically and genetically. Here we report on the isolation of a genomic locus that harbours the gene cluster ccoNOQP, and demonstrate that it encodes an alternative cbb3‐type cytochrome c oxidase. This oxidase has previously been shown to be specifically induced at low oxygen tensions, suggesting that its expression is controlled by an oxygen‐sensing mechanism. This view is corroborated by the observation that the ccoNOQP gene cluster is preceded by a gene that encodes an FNR homologue and that its promoter region contains an FNR‐binding motif. Biochemical and physiological analyses of a set of oxidase mutants revealed that, at least under the conditions tested, cytochromes aa3, bb3. and cbb3 make up the complete set of terminal oxidases in P. denitrificans. Proton‐translocation measurements of these oxidase mutants indicate that all three oxidase types have the capacity to pump protons. Previously, however, we have reported decreased H+/e coupling efficiencies of the cbb3‐type
The 4784V missense mutation of the dnaA5 and dnaA46 alleles confers a defect in ATP binding and thermoiability in initiation of Escherichia coli DNA replication
Authors: Carr Kevin M. , Kaguni Jon M.
Published: October 2006

The temperature‐sensitive dnaA5 and dnaA46 alleles each contain two missense mutations. These mutations have been separated and the resulting mutant proteins studied with regard to their role in initiation of DNA replication in vitro. Whereas the His‐252 to tyrosine substitution (H252Y) unique to the dnaA46 allele did not affect the activities of DnaA protein, the unique substitution of the dnaAS allele, Gly‐426 to serine (G426S), was reduced in its DNA‐binding affinity for oriC, the chromosomal origin. This suggests that the C‐terminal region of the DnaA protein is involved in DNA binding. The alanine‐to‐valine substitution at amino acid 184 (A184V) that is common to both of the alleles is responsible for the thermolabile defect and lag in DNA synthesis of these mutants. Mutant proteins bearing the common substitution were defective in ATP binding and were inactive in a replication system reconstituted with purified proteins. DnaK and GrpE protein activated these mutant proteins for replication and ATP binding; the latter was measured indirectly by the ATP‐dependent formation of a tryp‐sin‐resistant peptide. However, with this assay, the ATP‐binding affinity appeared to be reduced relative to wild‐type DnaA protein. Activation was by conversion of a self‐aggregate to the monomer, and also by a conformational alteration that correlated with ATP binding.
Dual regulation of the paraquat‐inducible gene pqi‐5 by SoxS and RpoS in Escherichia coli
Authors: Koh Young‐Sang , Roe Jung‐Hye
Published: October 2006

The pqi‐5 gene, producing a probable membrane protein of unknown function, has been reported to be a member of the soxRS regulon. The SoxRS‐dependent induction of pqi‐5 by paraquat occurs only during the exponential phase. The expression of pqi‐5 increased in the absence of paraquat during the stationary phase or under conditions of carbon or phosphate starvation. This increase was regulated at the transcriptional level by RpoS (σs), which recognized the second promoter (P2) approx. 5 nucleotides upstream from the promoter (P1) used at the exponential phase. Studies with a series of 5’deletions revealed that the paraquat‐responsive element resides between ‐52 and ‐42 nucleotides upstream from the P1 start site, whose nucleotide sequence matches closely to other SoxS‐binding sequences. The stationary‐phase induction required sequences up to position ‐42, which correspond to the 5’border of the putative ‐35 hexamer for the P2 promoter. The binding of the purified SoxS protein to the pqi‐5 promoter upstream sequences was demonstrated by gel mobility‐shift and DNase I protection assays. The transcription from P1 promoter by EσD was activated by purified SoxS in vitro, as was observed in vivo. The dual regulation of pqi‐5 by SoxS at the exponential phase and RpoS at the stationary phase is the first to be reported among the members of the soxRS regulon, suggesting that this gene might indeed play some role under stressful conditions.
Effect of O side‐chain length and composition on the virulence of Shigella flexneri 2a
Authors: Sandlin Robin C. , Goldberg Marcia B. , Maurelli Anthony T.
Published: October 2006

IcsA of Shigella flexneri is required for intercellular spread and is located in the outer membrane at one pole of the bacterium, where it catalyses the polymerization of host‐cell actin. The formation of the actin tail provides the force to move the bacterium in a unidirectional manner through the host‐cell cytoplasm. We have previously demonstrated that rough lipopolysaccharide (LPS) mutants of S. flexneri 2a are avirulent and cannot form plaques in tissue‐culture monolayers. This inability to form plaques is associated with non‐polar localization of IcsA and loss of host‐cell membrane‐protrusion formation ('fireworks'). To define the minimal LPS structure required for fireworks formation, we constructed a strain of S. flexneri (BS497) that contains a mutation in rfc, encoding the O side‐chain polymerase, and a strain, BS520, that possesses a defective O side‐chain ligase due to a mutation in rfaL. BS497 produces a LPS that consists of a core with one repeat unit of the O side‐chain, while BS520 produces a LPS consisting of a complete core with no O side‐chain. BS497 remained invasive but did not form fireworks or plaques in tissue‐culture monolayers and was negative in the Serény test. BS520 was invasive, generated reduced numbers of short fireworks, and made tiny plaques, but it was negative in the Serény test. Analysis of BS497 with anti‐lcsA antibody demonstrated that IcsA was distributed over the entire cell surface. The distribution of IcsA on the surface of BS520 was predominantly unipolar, with some trail‐back of IcsA label along the sides of the bacterium. A similar pattern was seen when infected monolayers were stained for polymerized actin. These results suggest that both the presence and the length of the O side‐chain are important in the proper localization or maintenance of IcsA at the pole which subsequently affects the ability to form actin tails and produce fireworks. This reduced ability to form actin tails and fireworks results in a decreased ability of Shigella to move into adjacent host cells. To determine if the sugar composition of the O side‐chain is important in the ability to form fireworks, the rfb region of S. flexneri2a was replaced with the rfb region from Escherichia coll serotype O8 or O25. Both hybrids were invasive, formed plaques, and gave positive Serény reactions. These results suggest that, unlike LPS length, the sugar composition of the O side‐chain is not a critical requirement for the proper localization of IcsA and efficient intercellular movement.
Vacuolar H+‐ATPase and weak base action in Dictyostelium
Authors: Davies L. , Farrar N. A. , Satre M. , Dottin R. P. , Gross J. D.
Published: October 2006

Amoebae of Dictyostelium discoideum release ammonia during development, and the accumulation of this weak base is believed to be responsible for inhibiting fruiting‐body formation and switching aggregates into migrating slugs. Exposure to weak bases can also inhibit aggregation and cell‐type specific gene expression. The pathway by which weak bases influence development is not understood. We show here that the development of a set of mutants defective in acidification of intracellular acidic compartments is abnormally sensitive to inhibition by weak bases. Moreover even in the absence of added weak bases these mutants are delayed in aggregation and have a protracted migratory phase. The same behaviour is observed in trans‐formants harbouring an antisense construct for one of the vacuolar H+‐ATPase subunits. These results support the idea that weak bases exert their effects by inhibiting acidification of an intracellular acidic compartment.
Interleukin‐21 Induces T‐cell Activation and Proinflammatory Cytokine Secretion in Rheumatoid Arthritis
Authors: Li J. , Shen W. , Kong K. , Liu Z.
Published: October 2006

Interleukin (IL)‐21 is a CD4+ T‐cell‐derived cytokine, which is involved in innate and adaptive immune response. In this study, we analysed IL‐21 receptor (IL‐21R) expression in peripheral blood and synovial fluid mononuclear cells, and investigated the role of IL‐21 in the induction of proinflammatory cytokine production by peripheral blood T cells (PB‐T) and synovial fluid T cells (SF‐T) from patients with rheumatoid arthritis (RA). Immunohistochemical staining demonstrated that IL‐21R‐positive cells were significantly increased in inflamed synovial tissues of RA patients compared with osteoarthritis (OA) and healthy controls. Flow cytometric analysis confirmed that IL‐21R was mainly expressed in freshly isolated CD4, CD8, B and NK cells from peripheral blood and synovial fluid, but decreased gradually in T cells 24 h after anti‐CD3 stimulation. PB‐ and SF‐T cells from RA patients were more responsive to IL‐21 when compared with controls. Importantly, isolated PB‐ or SF‐T cells from RA patients, when stimulated with IL‐21 and anti‐CD3 MoAb, secreted markedly higher levels of TNF‐α and IFN‐γ than controls. These data indicate that IL‐21R is overexpressed in the inflamed synovial membrane and in peripheral blood or synovial fluid leukocytes of RA patients, and that IL‐21 enhances local T‐cell activation, proliferation and proinflammatory cytokine secretion. Thus, blockade of IL‐21R signalling pathway may have a therapeutic potential in acute RA patients.