bluuebunny/crossref_metadata_2025 huggingface dataset:
Repression of lipopolysaccharide biosynthesis in Escherichia coli by an antisense RNA of Acetobacter methanolicus phage Acm1
Authors: Mamat U. , Rietschel E. Th. , Schmidt G.
Published: October 2006

Lysogenic Acetobacter methanolicus strains carrying the prophage Acm1 were found to be unable to synthesize both the capsutar polysaccharide (CPS) and the O‐specific side‐chain of lipopolysaccharide (LPS) and to represent rough variants of the host bacterium. A 262 bp DNA fragment of phage Acm1, obviously required for interference with LPS biosynthesis, was cloned and expressed in Escherichia coli Independently of the O‐type, transformation of various E. coli strains with the recombinant DNA resulted in a suppression of biosynthesis of the O‐specific chains. The DNA fragment of phage Acm1 contained three very short open reading frames of 21, 24, and 36 bp. However, attempts to express phage‐encoded peptides were not successful. Instead, the Acm1‐derived DNA fragment was shown to code for the synthesis of a trans‐acting RNA molecule of 97 nucleotides, designated lbi (LPS biosynthesis‐interfering) RNA. This RNA contains sequence complementarity to E. coli target RNA sequences and appears to have the ability to form intracellularly RNA hybrid duplexes with mRNA. The data presented in this study support the hypothesis that the phenotypic effect of conversion to rough‐type LPS is accompanied by the expression of an antisense RNA of phage Acm1.
Molecular genetics of a chromosomal locus involved in copper tolerance in Escherichia coli K‐12
Authors: Fong Sheik‐Tao , Camakaris James , Lee Barry T. O.
Published: October 2006

The cutA locus, presumably involved in copper tolerance in Escherichia coli, was characterized by a mutation leading to copper sensitivity. Copper‐accumulation measurements with radioactive 64Cu6+ showed increased uptake by cutA copper‐sensitive mutant cells, and reduced uptake when the cutA mutation was complemented in trans. The locus was mapped using complementation of the cutA mutant to partial copper tolerance with wild‐type chromosomal fragments. The 3.2 kb DNA region involved in cutA was sequenced and analysed, revealing three significant open reading frames, none of which had been previously published. The products of all three open reading frames were identified, when synthesized with the T7 phage promoter expression system, as polypeptides of about 50kDa, 24kDa, and 13kDa, consistent with the sizes predicted from the DNA sequences. The 50kDa and 24kDa polypeptides were found in the bacterial inner membrane, and the 13kDa polypeptide with the cytoplasmic fraction. In addition to being required for copper tolerance, cutA affects tolerance levels to zinC., nickel, cobalt and cadmium salts. Transcriptional fusions of cutA with the lux operon showed induction by copper, zinc, nickel, cobalt and, to a lesser extent, cadmium, manganese and silver salts.
The biogenesis of c‐type cytochromes in Escherichia coli requires a membrane‐bound protein, DipZ, with a protein disulphide isomerase‐like domain
Authors: Crooke Helen , Cole Jeff
Published: October 2006

A mutant of Escherichia coli K‐12, JCB606, which lacks all five c‐type cytochromes synthesized during anaerobic growth in the presence of nitrite or tri‐methylamine‐N‐oxide (TMAO), was totally defective in Nrf activity and also partially defective in TMAO reductase activity. The mutation in strain JCB606 was shown to affect expression of the tor operon, which contributes almost equally with the products of the dms operon to the rate of TMAO reduction by bacteria during anaerobic growth in the presence of TMAO. The mutation in strain JCB606, dipZ, was mapped by P1 transduction close to the mel operon at co‐ordinate 4425 on the E. coli chromosome, the gene order being nrf–fdhF–mel–dipZ–ampC. Recombinant plasmids that restored Nrf activity to test‐tube cultures of the mutant were isolated from a cosmid library. A 2.7 kb EcoRV–Smal fragment (co‐ordinates 4443 to 4446 kb on the physical map of the E. coli chromosome) was found potentially to encode three genes arranged in at least two operons. The second gene, dipZ, was sufficient to complement the JCB606 mutation. The translated DNA sequence predicts that DipZ is a 53kDa integral membrane protein with a 37kDa N‐terminal domain including at least six membrane‐spanning helices and a 16kDa carboxy‐terminal hydrophilic domain which includes a protein disulphide isomerase‐like motif. It is suggested that DipZ is essential for maintaining cytochrome c apoproteins in the correct conformations for the covalent attachment of haem groups to the appropriate pairs of cysteine residues.
Treponema pallidum in gel microdroplets: a novel strategy for investigation of treponemal molecular architecture
Authors: Cox David L. , Akins Darrin R. , Porcella Stephen F. , Norgard Michael V. , Radolf Justin D.
Published: October 2006

Controversy exists regarding the constituents and antigenic properties of the Treponema pallidum outer membrane; a major point of contention concerns the cellular location(s) of the spirochaete's lipoprotein immunogens. To address these issues and circumvent problems associated with prior efforts to localize treponemal surface antigens, we developed a novel strategy for investigating T. pallidum molecular architecture. Virulent treponemes were encapsulated in porous agarose beads (gel microdroplets) and then probed in the presence or absence of Triton X‐100. Intact., encapsulated treponemes were not labelled by monospecific antisera directed against four major T. pallidum lipoproteins or a candidate T. pailidum outer membrane protein (TpN50) with C‐terminal sequence homology to Escherichia coli OmpA or by human or rabbit syphilitic serum. Each of these immunologic reagents, however, labelled encapsulated treponemes co‐incubated with detergent. In contrast, antibodies generated against isolated T, pal‐lidum outer membranes labelled intact organisms and the pattern of fluorescence was consistent with the distribution of rare outer membrane proteins visualized by freeze‐fracture electron microscopy. In addition to providing strong evidence that the protein portions of treponemal lipoproteins are located within the periplasmic space, these studies have extended our understanding of the topographical relationships among T. pallidum cell envelope constituents. They also demonstrate the feasibility of generating antibodies against rare outer membrane proteins and detecting them on the surfaces of virulent treponemes.
Low‐molecular‐weight succinoglycan is predominantly produced by Rhizobium meliloti strains carrying a mutated ExoP protein characterized by a periplasmic N‐terminal domain and a missing C‐terminal domain
Authors: Becker Anke , Niehaus Karsten , Pühler Alfred
Published: October 2006

The membrane topology of the Rhizobium meliloti 2011 ExoP protein involved in polymerization and export of succinoglycan was analysed by translational fusions of lacZ and phoA reporter genes to the exoP gene. Based on this analysis, the ExoP protein could be divided into an N‐terminal domain mainly located in the periplasmic space and a C‐terminal domain located in the cytoplasm. Whereas the C‐terminal domain of ExoP is characterized by a potential nucleotide‐binding motif, the N‐terminal ExoP domain contains the sequence motif‘PX2pX4SPKX11GXMXG1′, which is also present in proteins involved in the determination of O‐antigen chain length. R. meliloti strains carrying mutated exoP* genes, exclusively encoding the N‐terminal ExoP domain, produced a reduced amount of succinoglycan. This reduction could be suppressed by a mutation in the regulatory gene exoR. The ratio of low‐molecular‐weight to high‐molecular‐weight succinoglycan was significantly increased in the exoP* mutant strain. In the exoP*lexoR mutant strain only low‐molecular‐weight succinoglycan could be detected. Based on sequence homologies and similar hydropathic profiles, the N‐terminal domain of ExoP was proposed to be a member of a protein family thought to be involved in polysaccharide chain‐length determination.
Activation of the transcriptional regulator XylR of Pseudomonas putida by release of repression between functional domains
Authors: Fernández Silvia , de Lorenzo Víctor , Pérez‐Martin José
Published: October 2006

In the presence of toluene, xylenes and other structural analogues, the regulatory protein XylR, of the family of transcriptional regulators which act in concert with the σ54 factor, activate the promoter Pu of the TOL (toluene degradation) plasmid pWW0 of Pseudomonas putida. Amino acid changes Val‐219‐Asp and Ala‐220‐Pro, introducing a proline kink at the hinge region between the N‐terminal A domain and the central portion of XylR, resulted in a semi‐constitutive phenotype which mimicked the activating effect of aromatic inducers. This phenotype was further exacerbated by inserting extra amino acid residues within the same inter‐domain region. A truncated XylR protein devoid of the signal‐receiving, amino‐terminal portion of the protein stimulated the cognate promoter Pu at high levels independently of inducer addition, both in Escherichia coli and in Pseudomonas putida. Replacement of the amino‐terminal domain by a heterologous peptide derived from the MS2 virus polymerase resulted in a hybrid protein still able to bind DNA to the same extent in vivo as XylR, but unable to stimulate transcription. These data indicate that a key event in the activation of XyIR by toluene/xylenes is the release of the repression caused by the A domain of the protein on surfaces located at the central domain of the regulator.
The genetic basis of colony opacity in Streptococcus pneumoniae: evidence for the effect of box elements on the frequency of phenotypic variation
Authors: Saluja Sunil K. , Weiser Jeffrey N.
Published: October 2006

Streptococcus pneumoniae undergoes spontaneous phase variation in colony morphology. Differences in colony opacity have previously been shown to correlate with differences in the ability of organisms to colonize the mucosal surface of the nasopharynx in an animal model. The genetic basis of opacity variation was identified in transformation experiments. A DNA library, from a strain that varies at high frequency, was screened to identify a single clone capable of transforming a transparent recipient strain which varies at low frequency to an opaque phenotype. Analysis of this opacity locus revealed two genes, glpD and glpF, with similarity to genes required for glycerol metabolism in other bacteria. Following the pneumococcal glpF, repetitive intergenic elements, boxes A and C, were identified. These stem‐loop‐forming elements were not present in the same locus of the recipient strain. Although not required for phase variation in colony opacity, the box element was necessary for expression of phase variation at high frequency. Introduction of the box elements during transformation affected colony morphology, possibly by altering expression of a putative regulatory gene downstream from the box element. Mutagenesis within this region confirmed the contribution of the putative regulatory gene to the expression of colony opacity. Growth characteristics of strains generated in this study provide additional evidence for an association of differences in cell wall autolysis and variation in colony opacity.
Genetic studies reveal that myristoylCoA:protein N‐myristoyltransferase is an essential enzyme in Candida albicans
Authors: Weinberg Robin A. , McWherter Charles A. , Freeman Sandra K. , Wood David C. , Gordon Jeffrey I. , Lee Stephen C.
Published: October 2006

MyristoylCoA:protein N‐myristoyltransferase (Nmt) catalyses the co‐transiational, covalent attachment of myristate (C14:0) to the amino‐terminal glycine residue of a number of eukaryotic proteins involved in cellular growth and signal transduction. The NMT1 gene is essential for vegetative growth of Saccharomyces cerevisiae. Studies were carried out to determine if Nmt is also essential for vegetative growth of the pathogenic fungus Candida albicans. A strain of C. albicans was constructed in which one copy of NMT was partially deleted and disrupted. A Gly‐447 — Asp mutation was Introduced into the second NMT allele. This mutation produced marked reductions in catalytic efficiency at 24 and 37° C, as judged by in vitro kinetic studies of the wild‐type and mutant enzymes which had been expressed in, and purified from, Escherichia coli. The growth characteristics of isogenic NMT/NMT, NMT/Δnmt, and nmtΔ/nmtG447D C. albicans strains were assessed under a variety of conditions. Only the nmtδ/nmtG447D strain required myristate for growth. This was true at both 24 and 37°C. Palmitate could not substitute for myristate. Incubation of nmtΔ/nmtG447D cells at 37° C in the absence of myristate resulted in cell death as observed by the inability to form colonies on media supplemented with 500 μM myristate. Studies in an immunosuppressed‐mouse model of C. albicans infection revealed that the NMT/Δnmt strain produced 100% lethality within 7 d after intravenous administration while the isogenic nmtΔ/nmtG447G strain produced no deaths even after 21 d. These observations establish that Nmt is essential for vegetative growth of C. albicans and suggest that Inhibitors of this acyltransferase may be therapeutically useful fungicidal agents.
Purification and characterization of LasD: a second staphylolytic proteinase produced by Pseudomonas aeruginosa
Authors: Park Sukjoon , Galloway D. R.
Published: October 2006

We have previously described studies of a 22 kDa active fragment of the LasA proteinase. In follow‐up studies of LasA, we have discovered the separate existence of a 23 kDa proteinase which shares many of the enzymatic properties of LasA, including the ability to lyse heat‐killed staphylococoi. However, this apparent serine proteinase, which we designate LasD, is distinct from the 22 kDa active LasA protein for the following reasons: (i) the N‐terminal sequence of LasD shares no homology with LasA or the LasA precursor sequence; (ii) Pseudomonas aeruginosa LasA mutant strains AD1825 and FRD2128 do not produce LasA yet produce LasD; and (iii) specific antibodies to each proteinase do not show any cross‐reactivity. LasD appears to be produced as a 30 kDa protein, which is possibly cleaved to produce a 23 kDa active fragment. The purified LasD fragment (23 kDa) shows strong staphylolytic activity only at higher pH conditions, while LasA exhibits staphylolytic activity over a broad pH range, in addition to their ability to cleave at internal diglycine sites, both the LasD and LasA endoproteinases efficiently cleave β‐casein.
Lipopolysaccharides of polymyxin B‐resistant mutants of Escherichia coii are extensively substituted by 2‐aminoethyl pyrophosphate and contain aminoarabinose in lipid A
Authors: Nummila Kim , Kilpeläinen IIkka , Zähringer Ulrich , Vaara Martti , Helander IIkka M.
Published: October 2006

Lipopolysaccharides (LPS) of two polymyxin‐resistant (pmr) mutants and the corresponding parent strain of Escherichia Coli were chemically analysed for composition and subjected to 31P‐NMR (nuclear magnetic resonance) for assessment of phosphate substitution. Whereas the saccharide portions, fatty acids, and phosphate contents were similar in wild‐type and pmr LPS, the latter contained two‐ to threefold higher amounts of 2‐aminoethanol. The pmr LPS also contained 4‐amino‐4‐deoxy‐l‐arabinopyranose (l‐Arap4N), which is normally not a component of E. coli LPS. This aminopentose has been assigned to be linked to the 4′‐phosphate of lipid A. Comparative 31P‐NMR analysis of the de‐O‐acylated LPS of the wild‐type and pmr strains revealed that phosphate groups of the pmr LPS were mainly (71‐79%) diphosphate diesters, which accounted for only 20% in the wild‐type LPS. Diphosphate monoesters were virtually nonexistent in the pmr LPS, whereas they accounted for 42% of all phosphates in wild‐type LPS. In the lipid A of the pmr strains, the 4′‐phosphate was to a significant degree (35%) substituted by l‐Arap4N, whereas in the wild‐type LPS the l‐ArapN was absent. In the pmr lipid A1 2‐aminoethanol was completely substituting the glycosidic pyrophosphate but not the glycosidic monophosphate, forming a diphosphate diester linkage at this position in 40% of lipid A molecules. In the wild‐type LPS the glycosidic position of lipid A carried mostly unsubstituted monophosphate and pyrophosphate. Thus the polymyxin resistance was shown to be associated, along with the esterification of the lipid A 4′‐monophosphate by aminoarabinose, with extensive esterification of diphosphates in LPS by 2‐aminoethanol.
Enhanced secretion through the Shigella flexneri Mxi‐Spa translocon leads to assembly of extracellular proteins into macromolecular structures
Authors: Parsot Claude , Ménard Robert , Gounon Pierre , Sansonetti Philippe J.
Published: October 2006

Genes required for entry of Shigella flexneri into epithelial cells in vitro are clustered in two adjacent loci, one of which encodes secretory proteins, the IpaA–D proteins, and the other their dedicated secretion apparatus, the Mxi–Spa translocon. Ipa secretion, which is induced upon contact of bacteria with epithelial cells, is prevented during growth in vitro. Here, we show that ipaB and ipaD mutations lead to enhanced secretion of a set of about 15 proteins. These extracellular proteins and some Ipas associate in organized structures consisting of extended sheets. Growth of the wild‐type strain in the presence of Congo red is shown to induce protein secretion through the Mxi–Spa translocon. Cultures grown to stationary phase in the presence of Congo red contain extracellular filaments whose composition and morphology are similar to those produced by the hyper‐secreting ipaB and ipaD mutants.
Isolation and characterization of genes that promote the expression of inositol transporter gene ITR1 in Saccharomyces cerevisiae
Authors: Nikawa J. , Hosaka K.
Published: October 2006

The expression of many genes of Saccharomyces cerevisiae, such as ITR1, is regulated by inositol and choline. In this work, a yeast strain has been constructed in which HIS3 expression is controlled by the ITR1 promoter. Using this strain, three genes were isolated which, when Introduced as multicopies, abolish the repression caused by inositol via the ITR1 promoter. Northern blot analysis revealed that two of these three genes, designated as DIE1 and DIE2, clearly increased the expression of ITR1. DIE2 is more effective for ITR1 expression than DIE1. Gene‐disruption experiments revealed that DIE1 was essential for the expression of ITR1 but that DIE2 was not. The sequence of the DIE1 gene was shown to be identical to that of INO2 (also called SCS1), which encodes a protein required for the expression of INO1. DIE2 is a new gene and is capable of encoding 525 amino acid residues with a calculated molecular weight of 61 789. Experiments involving lacZfusion genes showed that multicopy DIE2 resulted in an increase in the expression of both ITR1 and INO1. These results strongly suggest that the DIE1 and DIE2 gene products have an important regulatory function for gene expression of not only ITR1 but also INO1.
The Escherichia coli genes sspA and rnk can functionally replace the Pseudomonas aeruginosa alginate regulatory gene algR2
Authors: Schlictman David , Shankar Sandeep , Chakrabarty A. M.
Published: October 2006

The algR2 (also known as algQ) gene of Pseudomonas aeruginosa has previously been identified as being necessary for alginate production at 37°C. We have cloned two genes, from a cosmid library of Escherichia coli, which can restore mucoidy to an algR2 mutant of P. aeruginosa. The complementing regions of both cosmids were localized by subcloning restriction fragments. One of the E. coli genes identified here has not previously been described; we have named this gene rnk (regulator of nucleoside diphosphate kinase). It encodes a 14.9 kDa protein with no homo‐logy to any other protein. The other gene, sspA, is a regulator involved in stationary‐phase regulation in E. coli. Either gene will restore mucoidy to an algR2‐deficient strain of P. aeruginosa. AlgR2 has been shown to regulate at least two enzymes, succinyl‐CoA synthetase (Scs) and nucleoside diphosphate kinase (Ndk), which form a complex in P. aeruginosa. When we examined the ability of the E. coli analogues to regulate Ndk, we found that rnk but not sspA was able to restore Ndk activity to the P. aeruginosa algR2 mutant. Furthermore, rnk was able to restore growth of the algR2 mutant in the presence of Tween 20, which inhibits other Ndk‐like activities.
Genetic and molecular analysis of a regulatory region of the herbicide 2,4‐dichlorophenoxyacetate catabolic plasmid pJP4
Authors: You I.‐S. , Ghosal D.
Published: October 2006

In Alcaligenes eutrophus JMP134, pJP4 carries the genes coding for 2,4‐dichlorophenoxyacetate (2,4‐D) and 3‐chlorobenzoate (3‐Cba) degradation plus mercury resistance. The plasmid genes specifying 2,4‐D and 3‐Cba catabolism are organized in three operons: tfdA, tfdB, and tfdCDEF. Regulation of these operons by two unlinked genes, tfdR and tfdS, has been proposed. Physical and DNA sequence analyses revealed that the tfdR and tfdS genes were identical and were located within a longer inverted repeat of 1592bp. Similar stem‐loop structures were observed among other 2,4‐D plasmids. The tfdR gene is 888 bp long and capable of encoding a polypeptide of 32kDa. The deduced amino acid sequence of tfdR indicates that it is a member of the LysR‐type activators. Investigation of the regulation of the catabolic gene clusters through the construction of a pJP4 defined deletion mutant, pYG1010, which lacks a 4.2 kilobase Xbal fragment containing the inverted repeat region carrying the tfdR and tfdS regulatory genes, showed that Pseudomonas cepacia strains containing pYG1010 became 2,4‐D negative, but 3‐Cba positive. In vivo recombinants of pYG1010 and a cloned tfdS gene rescued the 2,4‐D phenotype, indicating that TfdS is a positive regulator of tfdA expression, but not for tfdCDEF expression.
The tagGH operon of Bacillus subtilis 168 encodes a two‐component ABC transporter involved in the metabolism of two wall teichoic acids
Authors: Lazarevic Vladimir , Karamata Dimitri
Published: October 2006

We report the nucleotide sequence and the characterization of the Bacillus subtilis tagGH operon. The latter is controlled by a σA‐dependent promoter and situated in the 308° chromosomal region which contains genes involved in teichoic acid biosynthesis. TagG is a hydrophobic 32.2 kDa protein which resembles integral membrane proteins belonging to polymerexport systems of Gram‐negative bacteria. Gene tagH encodes a 59.9 kDa protein whose N‐moiety contains the ATP‐binding motif and shares extensive homology with a number of ATP‐binding proteins, particularly with those associated with the transport of capsular polysaccharides and O‐antigens. That the tagGH operon is essential for cell growth was established by the failure to inactivate tagG and the 5′ ‐moiety of tagH by insertional mutagenesis. During limited tagGH expression, cells exhibited a cocoid morphology while their walls contained reduced amounts of phosphate as well as galactosamine. These observations, revealing impaired metabolism of both wall teichoic acids of B. subtilis 168, i.e. poly(glycerol phosphate), and poly(glucose galactosamine phosphate), combined with sequence homologies, suggest that TagG and TagH are involved in the translocation through the cytoplasmic membrane of the latter teichoic acids or their precursors.
Saturation of penicillin‐binding protein 1 by β‐lactam antibiotics in growing cells of Bacillus licheniformis
Authors: Lepage Sophie , Lakaye Bernard , Galleni Moreno , Thamm Iris , Crine Michel , Groslambert Sylvie , Frère Jean‐Marie
Published: October 2006

With the help of a new highly sensitive method allowing the quantification of free penicillin‐binding proteins (PBPs) and of an integrated mathematical model, the progressive saturation of PBP1 by various β‐lactam antibiotics in growing cells of Bacillus licheniformis was studied. Although the results confirmed PBP1 as a major lethal target for these compounds, they also underlined several weaknesses in our present understanding of this phenomenon. In growing cells, but not in resting cells, the penicillin target(s) appeared to be somewhat protected from the action of the inactivators. In vitro experiments indicated that amino acids, peptides and depsipeptides mimicking the peptide moiety of the nascent peptidoglycan significantly interfered with the acylation of PBP1 by the antibiotics. In addition, the level of PBP1 saturation at antibiotic concentrations corresponding to the minimum inhibitory concentrations was not constant, suggesting that additional, presently undiscovered, factors might be necessary to account for the experimental observations.
Repair, refold, recycle: how bacteria can deal with spontaneous and environmental damage to proteins
Authors: Visick Jonathan E. , Clarke Steven
Published: October 2006

Proteins, like DNA, are subject to various forms of damage that can render them non‐functional. Conformational changes and covalent chemical alterations occur spontaneously, and the rates of these reactions can be increased by environmental stresses such as heat, oxidative agents, or changes in pH or osmotic conditions. Although affected proteins can be replaced by de novo biosynthesis, cells – especially those subjected to stress or nutrient limitation – have developed mechanisms which can either restore damaged polypeptides to an active state or remove them. Such mechanisms can spare the biosynthetic capacity of the cell and ensure that the presence of non‐functional molecules does not disrupt cell physiology. Three major mechanisms, which operate in bacteria as well as eukaryotic organisms, have been described. First, chaperones not only assist in proper de novo folding of proteins but also provide an important means of restoring activity to conformationally damaged proteins. Second, enzymatic‘repair’systems exist to directly reverse certain forms of protein damage, including proline isomerization, methionine oxidation and the formation of isoaspartyl residues. Finally, proteolysis provides a‘last‐resort’means of dealing with abnormal proteins which cannot be repaired. Protein maintenance and repair may be of special importance for bacteria preparing to survive extended periods in stationary phase: both constitutive and induced mechanisms are utilized to permit survival despite greatly reduced protein synthesis.
Cloning, expression and functional analyses of the catabolite control protein CcpA from Bacillus megaterium
Authors: Hueck Christoph J. , Kraus Alexandra , Schmiedel Dagmar , Hillen Wolfgang
Published: October 2006

A mutant of Bacillus megaterium relieved from catabolite repression has been used to clone ccpA from B. megaterium by complementation. ccpA is the first gene of a presumed operon, in which it is followed by the motA homologue ORF1 and the motB homologue ORF2. The mutation maps in the 3′‐terminal region of ccpA, where an in‐frame duplication of 84 nucleotides located between two 9 bp direct repeats leads to an insertion of 28 amino acids near the C‐terminus of CcpA. An in‐frame deletion of 501 bp in ccpA exhibits the same phenotype as the 84 bp duplication. Deletion of ORF1 and ORF2 does not yield an apparent phenotype. A single‐copy ccpA::lacZ transcriptional fusion is constitutively expressed, independent of whether the growth medium triggers catabolite repression or not. The ccpA mutation leads to relief of catabolite repression exerted by glucose, fructose, mannitol, glucitol and glycerol, whereas only smaller effects were found with ribose, citrate and glutamate. The respective growth rates on these carbon sources are uniformly reduced to a generation time of about 90 min in the ccpA mutant. Catabolite repression of a plasmid‐encoded xylA::ccpA fusion is less efficient than that of a xylA::lacZ fusion in the same vector. Furthermore, overproduction of CcpA decreases catabolite repression of a singlecopy xylA::lacZ fusion approximately twofold. Thus, overexpression of CcpA may be counterproductive for catabolite repression, supporting the hypothesis that CcpA by itself may not bind sufficiently strongly to the cis‐active catabolite‐responsive element to exert catabolite repression.
Characterization of the gene encoding the immunodominant 35 kDa protein of Mycobacterium leprae
Authors: Winter Nathalie , Triccas James A. , Rivoire Becky , Pessolani Maria Cristina V. , Eiglmeier Karin , Lim Eng‐Mong , Hunter Shirley W. , Brennan Patrick J. , Britton Warwick J.
Published: October 2006

Analysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant antigen. The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane protein I). As the purified protein was not amenable to N‐terminal sequencing, partial proteolysis was used to establish the sequences of 21 peptides. A fragment of the 35 kDa protein‐encoding gene was amplified by the polymerase chain reaction from M. leprae chromosomal DNA with oligonucleotide primers derived from internal peptide sequences and the whole gene was subsequently isolated from a M. leprae cosmid library. The nucleotide sequence of the gene revealed an open reading frame of 307 amino acids containing most of the peptide sequences derived from the native 35 kDa protein. The calculated subunit mass was 33.7 kDa, but the native protein exists as a multimer of 950 kDa. Database searches revealed no identity between the 35 kDa antigen and known protein sequences. The gene was expressed in Mycobacterium smegmatis under the control of its own promoter or at a higher level using an‘up‐regulated’promoter derived from Mycobacterium fortuitum. The gene product reacted with monoclonal antibodies raised to the native protein. Using the bacterial alkaline phosphatase reporter system, we observed that the 35 kDa protein was unable to be exported across the membrane of recombinant M. smegmatis. The 35 kDa protein‐encoding gene is absent from members of the Mycobacterium tuberculosis complex, but homologous sequences were detected in Mycobacterium avium, Mycobacterium haemophilum and M. smegmatis. The avaibility of the recombinant 35 kDa protein will permit dissection of both antibody‐ and T‐cell‐mediated immune responses in leprosy patients.
Novel organization of the site‐specific integration and excision recombination functions of the Staphylococcus aureus serotype F virulence‐converting phages φ13 and φ42
Authors: Carroll David , Kehoe Michael A. , Cavanagh David , Coleman David C.
Published: October 2006

Functions required for site‐specific integration and excision of the Staphylococcus aureus serotype F virulence‐converting phages φ13 and φ42 were localized and characterized. Like other temperate phages, integration of φ13 and φ42 sequences was found to require the product of an int gene located close to the phage attP site. Both int genes are almost identical, express proteins possessing characteristic features of the Int (integrase) family of recombinases, but share very little homology with previously described int genes, including those of the serotype B S. aureus phages L54a and φ11. Nevertheless, all four S. aureus phages share an almost identical short sequence located immediately 5′ to these distinct int genes, suggesting a common mechanism of int gene regulation. Upstream from these common sequences, the sequences of φ13 and φ42 are quite distinct from each other, and from the corresponding regions of φ11 and L54a which encode the Xis proteins that are required with Int to mediate site‐specific excision of the latter phages. Surprisingly, φ13 and φ42 sequences encompassing the attP sites and int genes, but lacking either an adjacent or more distant phage excision protein gene, were sufficient to mediate site‐specific excision of integrated phage DNA sequences.
Identification of the ligand‐binding domain of the surface‐located fibrinogen receptor (clumping factor) of Staphylococcus aureus
Authors: McDevitt D. , Francois P , Vaudaux P. , Foster T. J.
Published: October 2006

The ability of Staphylococcus aureus to bind to fibrinogen and fibrin is believed to be an important factor in the initiation of foreign‐body and wound Infections. Recently, we reported the cloning and sequencing of the gene clfA encoding the fibrinogen receptor (clumping factor, ClfA) of S. aureus strain Newman and showed that the gene product was responsible for the clumping of bacteria in soluble fibrinogen and for the adherence of bacteria to solid‐phase fibrinogen. This was confirmed here by showing that antibodies raised against purified Region A inhibited both of these properties. Also, immunofluorescent microscopic analysis of wild‐type Newman and a clfA::Tn917 mutant of Newman with anti‐ClfA Region A sera confirmed that Region A is exposed on the bacterial cell surface. Furthermore, polystyrene beads coated with the Region A protein formed clumps in soluble fibrinogen showing that the ClfA protein alone is sufficient for the clumping phenotype. Western immunoblotting with anti‐ClfA Region A antibodies identified the native ClfA receptor as a 185 kDa protein that was released from the cell wall of S. aureus by lysostaphin treatment. A single extensive ligand‐binding site was located within Region A of the ClfA protein. Truncated ClfA proteins were expressed in Escherichia coli. Lysates of E. coli and proteins that had been purified by affinity chromatography were tested for (i) their ability to bind fibrinogen in Western ligand blotting experiments, (ii) for their ability to inhibit clumping of bacteria in fibrinogen solution and adherence of bacteria to solid‐phase fibrinogen, and (iii) for their ability to neutralize the blocking activity of anti‐ClfA Region A antibody. These tests allowed the ligand‐binding domain to be localized to a 218‐residue segment (residues 332‐550) within Region A.
The Mycobacterium leprae genome: systematic sequence analysis identifies key catabolic enzymes, ATP‐dependent transport systems and a novel polA locus associated with genomic variability
Authors: Fsihi Hafida , Cole Stewart T.
Published: October 2006

In the framework of the mycobacterial genome sequencing project, a continuous 37049bp sequence from the Mycobacterium leprae chromosome has been determined. Computer analysis revealed 10 complete open reading frames, and nine of their products show similarity to known proteins. Seven of these were identified as the enzyme isocitrate lyase, two P‐type ATPase cation transporters, two AMP‐binding proteins, the ribosomal protein S1, and DNA polymerase I. Interestingly, the polA gene, encoding DNA polymerase, is flanked by two inverted copies of a new class of the M. leprae specific repetitive sequence, RLEP, and this structure resembles a transposable element. A second copy of this element was found at another locus in the genome, but the two copies were not present in equal amounts and could not be found in all isolates of M. leprae. This is the first evidence for genomic variability in the leprosy bacillus and might ultimately be useful for developing a molecular test capable of distinguishing between strains of M. leprae.
Unique gene organization of thioredoxin and thioredoxin reductase in Mycobacterium leprae
Authors: Wieles Brigitte , van Soolingen Dick , Holmgren Arne , Offringa Rienk , Ottenhoff Tom , Thole Jelle
Published: October 2006

The thioredoxin system comprising thioredoxin (Trx), thioredoxin reductase (TR) and NADPH operates via redox‐active disulphides and provides electrons for a wide variety of different metabolic processes in prokaryotic and eukaryotic cells. Thioredoxin is also a general protein disulphide reductase involved in redox regulation. In bacteria, the Trx and TR proteins previously identified were encoded by separate genes (trxA and trxB). In this study, we report a novel genomic organization of TR and Trx in mycobacteria and show that at least three modes of organization of TR and Trx genes can exist within a single bacterial genus: (i) in the majority of mycobacterial strains the genes coding for TR and Trx are located on separate sites of the genome; (ii) interestingly, in all pathogenic Mycobacterium tuberculosis complex mycobacteria both genes are found on the same locus, overlapping in one nucleotide; (iii) in the pathogen Mycobacterium leprae, TR and Trx are encoded by a single gene. Sequence analysis of the M. leprae gene demonstrated that the N‐terminal part of the protein corresponds to TR and the C‐terminal part to Trx. A corresponding single protein product of approximately 49 kDa was detected in cell extracts of M. leprae. These findings demonstrate the very unusual phenomenon of a single gene coding for both the substrate (thioredoxin) and the enzyme (thioredoxin reductase), which seems to be unique to M. leprae.
Membrane topology and site‐specific mutagenesis of Pseudomonas aeruginosa porin OprD
Authors: Huang Honglin , Jeanteur Denis , Pattus Franc , Hancock Robert E. W.
Published: October 2006

Pseudomonas aeruginosa OprD is a 420‐amino‐acid protein that facilitates the uptake of basic amino acids, imipenem and gluconate across the outer membrane. OprD was the first specific porin that could be aligned with members of the non‐specific porin super‐family. Utilizing multiple alignments in conjugation with structure predictions and amphipathicity calculations, an OprD‐topology model was proposed. Sixteen β‐strands were predicted, connected by short loops at the periplasmic side. The eight external loops were of variable length but tended to be much longer than the periplasmic ones. Polymerase chain reaction (PCR)‐based site‐specific mutagenesis was performed to delete separately short stretches (4‐8 amino acid residues) from each of the predicted external loops. The mutants with deletions in the predicted external loops L1, L2, L5, L6, L7 and L8 were tolerated in both Escherichia coli and P. aeruginosa. The expressed mutant proteins maintained substantial resistance to trypsin treatment in the context of isolated outer membranes. Proteins with deletions in loops L1, L5, L6, L7 and L8 reconstituted similar imipenem supersusceptibility in a P. aeruginosa OprD::Ω background. The L2‐deletion mutant only partially reconstituted supersusceptibility, suggesting that loop L2 is involved in imipenem binding. These data were generally consistent with the topology model.
Exploring the Mycoplasma capricolum genome: a minimal cell reveals its physiology
Authors: Bork Peer , Ouzounis Christos , Casari Georg , Schneider Reinhard , Sander Chris , Dolan Maureen , Gilbert Walter , Gillevet Pat M.
Published: October 2006

We report on the analysis of 214 kb of the parasitic eubacterium Mycoplasma capricolum sequenced by genomic walking techniques. The 287 putative proteins detected to date represent about half of the estimated total number of 500 predicted for this organism. A large fraction of these (75%) can be assigned a likely function as a result of similarity searches. Several important features of the functional organization of this small genome are already apparent. Among these are (i) the expected relatively large number of enzymes involved in metabolic transport and activation, for efficient use of host cell nutrients; (ii) the presence of anabolic enzymes; (iii) the unexpected diversity of enzymes involved in DNA replication and repair: and (iv) a sizeable number of orthologues (82 so far) in Escherichia coli. This survey is beginning to provide a detailed view of how M. capricolum manages to maintain essential cellular processes with a genome much smaller than that of its bacterial relatives.
Molecular characterization of the eps gene cluster of Pseudomonas solanacearum and its transcriptional regulation at a single promoter
Authors: Huang Jianzhong , Schell Mark
Published: October 2006

Production of EPS I, an unusual exopolysaccharide virulence factor of the phytopathogen Pseudomonas solanacearum, requires the 18 kb eps gene cluster. DNA sequence analysis of the first seven genes of eps (epsAPBCDEF), subcellular localization of their products in maxicells, and phoA fusion analysis showed that: (i) epsA, epsB, epsE, and epsF encode exported or membrane‐associated proteins probably involved in polymerization and/or export of EPS I; (ii) epsC and epsD encode soluble enzymes probably involved in synthesis of sugar components of EPS I (N‐acetylgalactosaminuronic acid and possibly N‐acetyitrideoxygalactose, respectively); and (iii) epsP probably encodes a phosphatase involved in EPS I production in an unknown way. Non‐polar insertional mutagenesis showed that most, if not all, of these eps genes are absolutely required for production of EPS I. Using random eps::lacZ fusions and primer extension we located a transcription start site and promoter upstream of epsA. Analysis of a plasmid with this promoter fused to lacZ showed that a 140 bp regulatory region upstream of the eps transcription start site was sufficient for normal regulation of eps transcription by the multicomponent virulence gene regulatory network of P. solanacearum. Deletion of this eps promoter from a plasmid‐borne epsAPBCDE::lacZ fusion reduced its expression 10‐fold, indicating that this promoter alone is responsible for regulated transcription of an eps operon composed of at least epsAPBCDE. Analysis of genomic and plasmid‐borne eps::lacZ fusions suggested that most remaining eps genes are part of this same operon or, and this is less likely, comprise a second co‐ordinately regulated eps operon.
Importance of lipooligosaccharide structure in determining gonococcal resistance to hydrophobic antimicrobial agents resulting from the mtr efflux system
Authors: Lucas Claressa E. , Hagman Kayla E. , Levin James C. , Stein Daniel C. , Shafer William M.
Published: October 2006

Levels of gonococcal resistance to antimicrobial hydrophobic agents (HAs) are controlled by the mtr (multiple transferrable resistance) system, composed of the mtrRCDE genes. The mtrR gene encodes a transcriptional repressor that appears to regulate expression of the upstream and divergent mtrCDE operon. The mtrCDE genes encode membrane proteins analogous to the MexABOprK proteins of Pseudomonas aeruginosa that mediate export of structurally diverse antimicrobial agents. In this study we found that a single base pair deletion in a 13bp inverted repeat sequence within the mtrR promoter resulted in increased resistance of gonococci to both crystal violet (CV) and erythromycin (ERY) as well as to the more lipophilic non‐ionic detergent Triton X‐100 (TX‐100). However, this cross‐resistance was contingent on the production of a full‐length lipooligosaccharide (LOS) by the recipient strain used in transformation experiments. Introduction of this mutation (mtrR‐171) into three chemically distinct deep‐rough LOS mutants by transformation resulted in a fourfold increase in resistance to TX‐100 compared with a 160‐fold increase in an isogenic strain producing a full‐length LOS. However, both wild‐type and deep‐rough LOS strains exhibited an eightfold increase in resistance to CV and ERY as a result of the mtrR‐171 mutation. This suggests that gonococci have different LOS structural requirements for mtr‐mediated resistance to HAs that differ in their lipophilic properties. Evidence is presented that gonococci exclude HAs by an energy‐dependent efflux process mediated by the mtr system.
Structural requirements for the glycolipid receptor of human uropathogenic Escherichia coli
Authors: Striker Robert , Nilsson Ulf , Stonecipher Andrea , Magnusson Göran , Hultgren Scott J.
Published: October 2006

The binding of uropathogenic Escherichia coli to the globo series of glycolipids via P pili is a critical step in the infectious process that is mediated by a human‐specific PapG adhesin. Three classes of PapG adhesins exist with different binding specificities to Galα4Gal‐containing glycolipids. The structural basis for PapG recognition of the human glycolipid receptor globoside was investigated by using soluble saccharide analogues as inhibitors of bacterial haemagglutination. The minimum binding epitope was confirmed as the Galα4Gal moiety, but parts of the GalNAcβ and glucose residues, which flank the Galα4Gal in globoside (GbO4), were also shown to be important for strong binding. Furthermore, the same five hydroxyl groups of Galα4Gal in globotriasyl ceramide that were recognized by a previously characterized PapG variant were also recognized by the human‐specific PapG in binding the GbO4 that dominates In the human kidney. Saccharide analogues that blocked haemagglutination also blocked the adherence of human uropathogenic E. coli to human kidney sections. Knowledge of the molecular details of the PapG‐GbO4 interaction will make it possible to design antiadherence therapeutics.
Screening of stable proteins in an extreme thermophile, Thermus thermophilus
Authors: Tamakoshi Masatada , Yamagishi Akihiko , Oshima Tairo
Published: October 2006

The leuB gene codes for 3‐isopropylmalate dehydrogenase of the leucine biosynthetic pathway in an extreme thermophile, Thermus thermophilus. The leuB gene of the thermophile was replaced with a temperature‐sensitive chimeric leuB gene. The resultant transformant was adapted to high temperature, a thermostable mutant strain being obtained. A single base substitution that replaces isoleucine at 93 with leucine was found in the chimeric leuB gene of the thermostable mutant. The resultant amino acid residue coincided with the corresponding residue of the T. thermophilus enzyme. It was confirmed that the mutant enzyme is more stable than the original chimeric enzyme. This system can be used to produce stabilized mutants of other enzymes without structural knowledge of them.
Hypothesis: chromosome separation in Escherichia coli involves autocatalytic gene expression, transertion and membrane‐domain formation
Authors: Norris Vic
Published: October 2006

To explain how daughter chromosomes are separated into discrete nucleoids and why chromosomes are partitioned with pole preferences, I propose that differential gene expression occurs during DNA replication in Escherichia coli. This differential gene expression means that the daughter chromosomes have different patterns of gene expression and that cell division is not a simple process of binary fission. Differential gene expression arises from autocatalytic gene expression and creates a separate proteolipid domain around each developing chromosome via the coupled transcription‐translation–insertion of proteins into membranes (transertion). As these domains are immiscible, daughter chromosomes are simultaneously replicated and separated into discrete nucleoids. I also propose that the partitioning relationship between chromosome age and cell age arises because the poles of cells have a proteolipid composition that favours transertion from one nucleoid rather than from the other. This hypothesis forms part of an ensemble of related hypotheses which attempt to explain cell division, differentiation and wall growth in bacteria in terms of the physical properties and interactions of the principal constituents of cells.