bluuebunny/crossref_metadata_2025 huggingface dataset:
Prophages and bacterial genomics: what have we learned so far?
Authors: Casjens Sherwood
Published: December 2004

Epigraph

There is something fascinating about science. One gets such wholesale returns of conjecture out of such a trifling investment of fact.

Mark Twain 1883

 Life on the Mississippi

Summary Bacterial genome nucleotide sequences are being completed at a rapid and increasing rate. Integrated virus genomes (prophages) are common in such genomes. Fifty‐one of the 82 such genomes published to date carry prophages, and these contain 230 recognizable putative prophages. Prophages can constitute as much as 10–20% of a bacterium's genome and are major contributors to differences between individuals within species. Many of these prophages appear to be defective and are in a state of mutational decay. Prophages, including defective ones, can contribute important biological properties to their bacterial hosts. Therefore, if we are to comprehend bacterial genomes fully, it is essential that we are able to recognize accurately and understand their prophages from nucleotide sequence analysis. Analysis of the evolution of prophages can shed light on the evolution of both bacteriophages and their hosts. Comparison of the Rac prophages in the sequenced genomes of three Escherichia coli strains and the Pnm prophages in two Neisseria meningitidis strains suggests that some prophages can lie in residence for very long times, perhaps millions of years, and that recombination events have occurred between related prophages that reside at different locations in a bacterium's genome. In addition, many genes in defective prophages remain functional, so a significant portion of the temperate bacteriophage gene pool resides in prophages.
The siderophore system is essential for viability of Aspergillus nidulans: functional analysis of two genes encoding l‐ornithine N 5‐monooxygenase (sidA) and a non‐ribosomal peptide synthetase (sidC)
Authors: Eisendle Martin , Oberegger Harald , Zadra Ivo , Haas Hubertus
Published: December 2004

The filamentous ascomycete A. nidulans produces two major siderophores: it excretes triacetylfusarinine C to capture iron and contains ferricrocin intracellularly. In this study we report the characterization of two siderophore biosynthetic genes, sidA encoding l‐ornithine N5‐monooxygenase and sidC encoding a non‐ribosomal peptide synthetase respectively. Disruption of sidC eliminated synthesis of ferricrocin and deletion of sidA completely blocked siderophore biosynthesis. Siderophore‐deficient strains were unable to grow, unless the growth medium was supplemented with siderophores, suggesting that the siderophore system is the major iron assimilatory system of A. nidulans during both iron depleted and iron‐replete conditions. Partial restoration of the growth of siderophore‐deficient mutants by high concentrations of Fe2+ (but not Fe3+) indicates the presence of an additional ferrous transport system and the absence of an efficient reductive iron assmilatory system. Uptake studies demonstrated that TAFC‐bound iron is transferred to cellular ferricrocin whereas ferricrocin is stored after uptake. The siderophore‐deficient mutant was able to synthesize ferricrocin from triacetylfusarinine C. Ferricrocin‐deficiency caused an increased intracellular labile iron pool, upregulation of antioxidative enzymes and elevated sensitivity to the redox cycler paraquat. This indicates that the lack of this cellular iron storage compound causes oxidative stress. Moreover, ferricrocin biosynthesis was found to be crucial for efficient conidiation.
The Pseudomonas syringae type III‐secreted protein HopPtoD2 possesses protein tyrosine phosphatase activity and suppresses programmed cell death in plants
Authors: Espinosa Avelina , Guo Ming , Tam Vincent C. , Fu Zheng Qing , Alfano James R.
Published: December 2004

The bacterial plant pathogen Pseudomonas syringae possesses a type III protein secretion system that delivers many virulence proteins into plant cells. A subset of these proteins (called Avr proteins) is recognized by the plant's innate immune system and triggers defences. One defence‐associated response is the hypersensitive response (HR), a programmed cell death (PCD) of plant tissue. We have previously identified HopPtoD2 as a type III secreted protein from P. s. pv. tomato DC3000. Sequence analysis revealed that an N‐terminal domain shared homology with AvrPphD and a C‐terminal domain was similar to protein tyrosine phosphatases (PTPs). We demonstrated that purified HopPtoD2 possessed PTP activity and this activity required a conserved catalytic Cys residue (Cys378). Interestingly, HopPtoD2 was capable of suppressing the HR elicited by an avirulent P. syringae strain on Nicotiana benthamiana. HopPtoD2 derivatives that lacked Cys378 no longer suppressed the HR indicating that HR suppression required PTP activity. A constitutively active MAPK kinase, called NtMEK2DD, is capable of eliciting an HR‐like cell death when transiently expressed in tobacco. When NtMEK2DD and HopPtoD2 were co‐delivered into plant cells, the HR was suppressed indicating that HopPtoD2 acts downstream of NtMEK2DD. DC3000 hopPtoD2 mutants were slightly reduced in their ability to multiply in planta and displayed an enhanced ability to elicit an HR. The identification of HopPtoD2 as a PTP and a PCD suppressor suggests that the inactivation of MAPK pathways is a virulence strategy utilized by bacterial plant pathogens.
A translocated protein tyrosine phosphatase of Pseudomonas syringae pv. tomato DC3000 modulates plant defence response to infection
Authors: Bretz James R. , Mock Norton M. , Charity James C. , Zeyad Syed , Baker C. Jacyn , Hutcheson Steven W.
Published: December 2004

Pseudomonas syringae strains translocate effector proteins into host cells via the hrp‐encoded type III protein secretion system (TTSS) to facilitate pathogenesis in susceptible plants. However, the mechanisms by which pathogenesis is favoured by these effectors are not well understood. Individual strains express multiple effectors with apparently distinct activities that are co‐ordinately regulated by the alternative sigma factor HrpL. Genes for several effectors were identified in the P. syringae pv. tomato DC3000 genome using a promoter trap assay to identify HrpL‐dependent promoters. In addition to orthologues of avrPphE and hrpW, an unusual allele of avrPphD was detected that carried an IS52 insertion. Using this avrPphD::IS52 allele as a probe, a wild‐type allele of avrPphD, hopPtoD1, and a chimeric homologue were identified in the DC3000 genome. This chimeric homologue, identified as HopPtoD2 in the annotated DC3000 genome, consisted of an amino terminal secretion domain similar to that of AvrPphD fused to a potential protein tyrosine phosphatase domain. Culture filtrates of strains expressing HopPtoD2 were able to dephosphorylate pNPP and two phosphotyrosine peptides. HopPtoD2 was shown to be translocated into Arabidopsis thaliana cells via the hrp‐encoded TTSS. A ΔhopPtoD2 mutant of DC3000 exhibited strongly reduced virulence in Arabidopsis thaliana. Ectopic expression of hopPtoD2 in P. syringae Psy61 that lacks a native hopPtoD2 orthologue delayed the development of several defence‐associated responses including programmed cell death, active oxygen production and transcription of the pathogenesis‐related gene PR1. The results indicate that HopPtoD2 is a translocated effector with protein tyrosine phosphatase activity that modulates plant defence responses.
Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and break down existing biofilms
Authors: Kuiper Irene , Lagendijk Ellen L. , Pickford Russell , Derrick Jeremy P. , Lamers Gerda E. M. , Thomas‐Oates Jane E. , Lugtenberg Ben J. J. , Bloemberg Guido V.
Published: December 2004

Pseudomonas putida strain PCL1445 was isolated from roots of plants, grown on a site polluted with polycyclic aromatic hydrocarbons. PCL1445 produces biosurfactant activity at the end of the exponential growth phase. High‐performance liquid chromatography (HPLC) analysis of supernatant extracts of PCL1445 showed two peaks with surface‐tension reducing activity, tentatively assigned as biosurfactants putisolvin I and putisolvin II and was followed by structural analyses. A transposon mutant of PCL1445, strain PCL1436, which lacks the two surface‐active peaks appeared to be mutated in an open reading frame (ORF) with amino acid homology to various lipopeptide synthetases. Structural analyses of the two biosurfactants of PCL1445 revealed that both are novel cyclic lipodepsipeptides with a hexanoic lipid chain connected to the N‐terminus of a 12‐amino‐acid peptide moiety, in which the C‐terminal carboxylic acid group forms an ester with the hydroxyl side‐chain of Ser9. The difference between the two structures is located in the second amino acid from the C‐terminus, being valine for putisolvin I, and leucine/isoleucine for putisolvin II. We show that these novel compounds lower the surface tension and influence the biofilm development on polyvinyl chloride (PVC). Biofilm formation of the bio‐synthetic mutant PCL1436 was strongly increased containing more cells, which formed aggregates earlier as compared with wild‐type PCL1445 biofilms. Using purified putisolvin I and II it was shown that biofilm formation of different Pseudomonas strains was inhibited and most interestingly, that both putisolvins are also able to break down existing Pseudomonas biofilms.
Global regulation of virulence and the stress response by CsrA in the highly adapted human gastric pathogen Helicobacter pylori
Authors: Barnard Faye M. , Loughlin Michael F. , Fainberg Hernan P. , Messenger Michael P. , Ussery David W. , Williams Paul , Jenks Peter J.
Published: December 2004

Although successful and persistent colonization of the gastric mucosa depends on the ability to respond to changing environmental conditions and co‐ordinate the expression of virulence factors during the course of infection, Helicobacter pylori possesses relatively few transcriptional regulators. We therefore investigated the contribution of the regulatory protein CsrA to global gene regulation in this important human pathogen. CsrA was necessary for full motility and survival of H. pylori under conditions of oxidative stress. Loss of csrA expression deregulated the oxidant‐induced transcriptional responses of napA and ahpC, the acid induction of napA, cagA, vacA, the urease operon, and fur, as well as the heat shock responses of napA, groESL and hspR. Although the level of napA transcript was higher in the csrA mutant, its stability was similar in the wild‐type and mutant strains, and less NapA protein was produced in the mutant strain. Finally, H. pylori strains deficient in the production of CsrA were significantly attenuated for virulence in a mouse model of infection. This work provides evidence that CsrA has a broad role in regulating the physiology of H. pylori in response to environmental stimuli, and may be important in facilitating adaptation to the different environments encountered during colonization of the gastric mucosa. Furthermore, CsrA appears to mediate its effects in H. pylori at the post‐transcriptional level by influencing the processing and translation of target transcripts, with minimal effect on the stability of the target mRNAs.
Is modification sufficient to protect a bacterial chromosome from a resident restriction endonuclease?
Authors: Makovets Svetlana , Powell Lynn M. , Titheradge Annette J. B. , Blakely Garry W. , Murray Noreen E.
Published: December 2004

It has been generally accepted that DNA modification protects the chromosome of a bacterium encoding a restriction and modification system. But, when target sequences within the chromosome of one such bacterium (Escherichia coli K‐12) are unmodified, the cell does not destroy its own DNA; instead, ClpXP inactivates the nuclease, and restriction is said to be alleviated. Thus, the resident chromosome is recognized as ‘self’ rather than ‘foreign’ even in the absence of modification. We now provide evidence that restriction alleviation may be a characteristic of Type I restriction–modification systems, and that it can be achieved by different mechanisms. Our experiments support disassembly of active endonuclease complexes as a potential mechanism. We identify amino acid substitutions in a restriction endonuclease, which impair restriction alleviation in response to treatment with a mutagen, and demonstrate that restriction alleviation serves to protect the chromosome even in the absence of mutagenic treatment. In the absence of efficient restriction alleviation, a Type I restriction enzyme cleaves host DNA and, under these conditions, homologous recombination maintains the integrity of the bacterial chromosome.
Aberrant cardiolipin metabolism in the yeast taz1 mutant: a model for Barth syndrome
Authors: Gu Zhiming , Valianpour Fredoen , Chen Shuliang , Vaz Frederic M. , Hakkaart Gertjan A. , Wanders Ronald J. A. , Greenberg Miriam L.
Published: December 2004

In eukaryotic cells, the acyl species of the phospholipid cardiolipin (CL) are more highly unsaturated than those of the other membrane phospholipids. Defective acylation of CL with unsaturated fatty acids and decreased total CL are associated with Barth syndrome, an X‐linked cardio‐ and skeletal myopathy attributed to a defect in the gene G4.5 (also known as tafazzin). We constructed a yeast mutant (taz1) containing a null mutation in the homologue of the human G4.5 gene. The yeast taz1Δ mutant was temperature sensitive for growth in ethanol as sole carbon source, but grew normally on glucose or glycerol plus ethanol. Total CL content was reduced in the taz1Δ mutant, and monolyso‐CL accumulated. The predominant CL acyl species found in wild‐type cells, C18:1 and C16:1, were markedly reduced in the mutant, whereas CL molecules containing saturated fatty acids were present. Interestingly, CL synthesis increased in the mutant, whereas expression of the CL structural genes CRD1 and PGS1 did not, suggesting that de novo biosynthetic enzyme activities are regulated by CL acylation. These results indicate that the taz1Δ mutant is an excellent genetic tool for the study of CL remodelling and may serve as a model system for the study of Barth syndrome.
EthR, a repressor of the TetR/CamR family implicated in ethionamide resistance in mycobacteria, octamerizes cooperatively on its operator
Authors: Engohang‐Ndong Jean , Baillat David , Aumercier Marc , Bellefontaine Flore , Besra Gurdyal S. , Locht Camille , Baulard Alain R.
Published: December 2004

Ethionamide (ETH) is an important second‐line antitubercular drug used for the treatment of patients infected with multidrug‐resistant Mycobacterium tuberculosis. Although ETH is a structural analogue of isoniazid, only little cross‐resistance to these two drugs is observed among clinical isolates. Both isoniazid and ETH are pro‐drugs that need to be activated by mycobacterial enzymes to exert their antimicrobial activity. We have recently identified two M. tuberculosis genes, Rv3854c (ethA) and Rv3855 (ethR), involved in resistance to ETH. ethA encodes a protein that belongs to the Flavin‐containing monooxygenase family catalysing the activation of ETH. We show here that ethR, which encodes a repressor belonging to the TetR/CamR family of transcriptional regulators, negatively regulates the expression of ethA. By the insertion of the ethA promoter region upstream of the lacZ reporter gene, overexpression of ethR in trans was found to cause a strong inhibition of ethA expression, independently of the presence of ETH in the culture media. Electrophoretic mobility shift assays indicated that EthR interacts directly with the ethA promoter region. This interaction was confirmed by DNA footprinting analysis, which, in addition, identified the EthR‐binding region. Unlike other TetR/CamR members, which typically bind 15 bp operators, EthR recognises an unusually long 55 bp region suggesting multimerization of the repressor on its operator. Identification by primer‐extension of the ethA transcriptional start site indicated that it is located within the EthR‐binding region. Taken together, bacterial two‐hybrid experiments and gel filtration assays suggested a dimerization of EthR in the absence of its operator. In contrast, surface plasmon resonance analyses showed that eight EthR molecules bind cooperatively to the 55 bp operator, which represents a novel repression mechanism for a TetR/CamR member.
The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage
Authors: Pullinger Gillian D. , Bevir Thomas , Lax Alistair J.
Published: December 2004

Toxigenic strains of Pasteurella multocida produce a 146 kDa toxin (PMT) that acts as a potent mitogen. Sequence analysis of the structural gene for PMT, toxA, previously suggested it was horizontally acquired, because it had a low G + C content relative to the P. multocida genome. To address this, the sequence of DNA flanking toxA was determined. The sequence analysis showed the presence of homologues to bacteriophage tail protein genes and a bacteriophage antirepressor, suggesting that the toxin gene resides within a prophage. In addition to phage genes, the toxA flanking DNA contained a homologue of a restriction/modification system that was shown to be functional. The presence of a bacteriophage was demonstrated in spent medium from toxigenic P. multocida isolates. Its production was increased by mitomycin C addition, a treatment that is known to induce the lytic cycle of many temperate bacteriophages. The genomes of bacteriophages from three different toxigenic P. multocida strains had similar but not identical restriction profiles, and were approximately 45–50 kb in length. The prophages from two of these had integrated at the same site in the chromosome, in a tRNA gene. Southern blot analysis confirmed that these bacteriophages contained the toxA gene.
Restricting restriction
Authors: Bickle Thomas A.
Published: December 2004

Systems biology is a new, fashionable and well‐funded discipline, which to quote from a recent review aims to ‘examine the structure and dynamics of cellular and organismal function, rather than the characteristics of isolated parts of a cell or organism…’ (Kitano, H. (2002) Science 295:1662–1664). Systems biology will do this by profiting from the vast amounts of biological information that are available in the genomics era and make extensive use of computer modelling. But: ‘many breakthroughs in experimental devices, advanced software and analytical methods are required before the achievements of system biology can live up to their much‐touted potential’. This edition of Molecular Microbiology contains a paper that is the product of traditional experimental biology but which could serve as a test case for systems biology. The paper shows how bacteria integrate such disparate subsystems as DNA restriction, homologous recombination and regulated proteolysis to protect their chromosomes from degradation. When systems biology can predict this level of choreography, it will be a mature discipline.
Global impact of mature biofilm lifestyle on Escherichia coli K‐12 gene expression
Authors: Beloin Christophe , Valle Jaione , Latour‐Lambert Patricia , Faure Philippe , Kzreminski Mickaël , Balestrino Damien , Haagensen Janus A. J. , Molin Søren , Prensier Gérard , Arbeille Brigitte , Ghigo Jean‐Marc
Published: December 2004

The formation of biofilm results in a major lifestyle switch that is thought to affect the expression of multiple genes and operons. We used DNA arrays to study the global effect of biofilm formation on gene expression in mature Escherichia coli K‐12 biofilm. We show that, when biofilm is compared with the exponential growth phase, 1.9% of the genes showed a consistent up‐ or downregulation by a factor greater than two, and that 10% of the E. coli genome is significantly differentially expressed. The functions of the genes induced in these conditions correspond to stress response as well as energy production, envelope biogenesis and unknown functions. We provide evidence that the expression of stress envelope response genes, such as the psp operon or elements of the cpx and rpoE pathways, is a general feature of E. coli mature biofilms. We also compared biofilm with the stationary growth phase and showed that the biofilm lifestyle, although sharing similarities with the stationary growth phase, triggers the expression of specific sets of genes. Using gene disruption of 54 of the most biofilm‐induced genes followed by a detailed phenotypic study, we validated the biological relevance of our analysis and showed that 20 of these genes are required for the formation of mature biofilm. This group includes 11 genes of previously unknown function. These results constitute a comprehensive analysis of the global transcriptional response triggered in mature E. coli biofilms and provide insights into its physiological signature.
Multiple stress signal integration in the regulation of the complex σS‐dependent csiD‐ygaF‐gabDTP operon in Escherichia coli
Authors: Metzner Martin , Germer Jens , Hengge Regine
Published: December 2004

The csiD‐ygaF‐gabDTP region in the Escherichia coli genome represents a cluster of σS‐controlled genes. Here, we investigated promoter structures, sigma factor dependencies, potential co‐regulation and environmental regulatory patterns for all of these genes. We find that this region constitutes a complex operon with expression being controlled by three differentially regulated promoters: (i) csiDp, which affects the expression of all five genes, is cAMP‐CRP/σS‐dependent and activated exclusively upon carbon starvation and stationary phase; (ii) gabDp1, which is σS‐dependent and exhibits multiple stress induction like σS itself; and (iii) gabDp2[previously suggested by Schneider, B.L., Ruback, S., Kiupakis, A.K., Kasbarian, H., Pybus, C., and Reitzer, L. (2002) J. Bacteriol. 184: 6976–6986], which appears to be Nac/σ70‐controlled and to respond to poor nitrogen sources. In addition, we identify a novel repressor, CsiR, which modulates csiDp activity in a temporal manner during early stationary phase. Finally, we propose a physiological  role  for σS‐controlled  GabT/D‐mediated γ‐aminobutyrate (GABA) catabolism and glutamate accumulation in general stress adaptation. This physiological role is reflected by the activation of the operon‐internal gabDp1 promoter under the different conditions that also induce σS, which include shifts to acidic pH or high osmolarity as well as starvation or stationary phase.
Application of AgaR repressor and dominant repressor variants for verification of a gene cluster involved in N‐acetylgalactosamine metabolism in Escherichia coli K‐12
Authors: Ray W. Keith , Larson Timothy J.
Published: December 2004

The agaZVWEFASYBCDI gene cluster encodes the phosphotransferase systems and enzymes responsible for the uptake and metabolism of N‐acetylgalactosamine and galactosamine in Escherichia coli. In some strains of E. coli, particularly the common K‐12 strain, a portion of this cluster is missing because of a site‐specific recombination event that occurred between sites in agaW and agaA. Strains that have undergone this recombination event have lost the ability to utilize either N‐acetylgalactosamine or galactosamine as sole sources of carbon. Divergently transcribed from this gene cluster is the gene agaR encoding a transcriptional repressor belonging to the DeoR/GlpR family of transcriptional regulators. Promoters upstream of agaR, agaZ and agaS were characterized. All three promoters had elevated activity in the presence of N‐acetylgalactosamine or galactosamine, were regulated in vivo by AgaR and possessed specific DNA‐binding sites for AgaR upstream from the start sites of transcription as determined by DNase I footprinting. In vivo analysis and DNase I footprinting indicated that the promoter specific for agaZ also requires activation by cAMP‐CRP. Previous work with GlpR and other members of the DeoR/GlpR family have identified highly conserved amino acid residues that function in DNA‐binding or response to inducer. These residues of AgaR were targeted for site‐directed mutagenesis and yielded variants of AgaR that were either negatively dominant or non‐inducible. The apparent ability to produce negatively dominant and non‐inducible variants of proteins of the DeoR/GlpR family of currently unknown function will likely facilitate screening for function.
The HCC‐domain of botulinum neurotoxins A and B exhibits a singular ganglioside binding site displaying serotype specific carbohydrate interaction
Authors: Rummel Andreas , Mahrhold Stefan , Bigalke Hans , Binz Thomas
Published: December 2004

Tetanus and botulinum neurotoxins selectively invade neurons following binding to complex gangliosides. Recent biochemical experiments demonstrate that two ganglioside binding sites within the tetanus neurotoxin HC‐fragment, originally identified in crystallographic studies to bind lactose or sialic acid, are required for productive binding to target cells. Here, we determine by mass spectroscopy studies that the HC‐fragment of botulinum neurotoxins A and B bind only one molecule of ganglioside GT1b. Mutations made in the presumed ganglioside binding site of botulinum neurotoxin A and B abolished the formation  of  these  HC‐fragment/ganglioside  complexes, and drastically diminished binding to neuronal membranes and isolated GT1b. Furthermore, correspondingly mutated full‐length neurotoxins exhibit significantly reduced neurotoxicity, thus identifying a single ganglioside binding site within the carboxyl‐terminal half of the HC‐fragment of botulinum neurotoxins A and B. These binding cavities are defined by the conserved peptide motif H…SXWY…G. The roles of tyrosine and histidine in botulinum neurotoxins A and B in ganglioside binding differ from those in the analogous tetanus neurotoxin lactose site. Hence, these findings provide valuable information for the rational design of potent botulinum neurotoxin binding inhibitors.
MurAA, catalysing the first committed step in peptidoglycan biosynthesis, is a target of Clp‐dependent proteolysis in Bacillus subtilis
Authors: Kock Holger , Gerth Ulf , Hecker Michael
Published: December 2004

The carboxyvinyl transfer from phosphoenolpyruvate to UDP‐N‐acetylglucosamine is the first committed step in the pathway of peptidoglycan formation. This crucial reaction for bacterial cell growth is catalysed by the MurA enzymes. Gram‐negative bacteria carry one murA gene, whereas in a subgroup of Gram‐positive bacteria two separate paralogues, MurAA and MurAB, exist. This study provides evidence that in the Gram‐positive bacterium Bacillus subtilis, the MurAA protein is specifically degraded by the ClpCP protease. This Clp‐dependent degradation is especially enhanced upon entry into stationary phase, thus ensuring an immediate growth arrest due to stalled murein biosynthesis. The MurAA protein can therefore be addressed as a target of Clp‐dependent regulatory proteolysis such as the transcriptional regulators CtsR, ComK, Spx in B. subtilis, CtrA in Caulobacter crescentus or RpoS in Escherichia coli. Taking into account all other known regulatory targets of ATP‐dependent proteases, MurAA of B. subtilis represents the first example of a metabolic enzyme which is a unique regulatory substrate of Clp‐dependent proteolysis. Its function as a regulatory metabolic checkpoint resembles that of homoserine trans‐succinylase (MetA) in E. coli which is similarly ATP‐dependently degraded.
Structural studies of Streptococcus pneumoniae EPSP synthase in unliganded state, tetrahedral intermediate‐bound state and S3P‐GLP‐bound state
Authors: Park HaJeung , Hilsenbeck Jacqueline L. , Kim Hak Jun , Shuttleworth Wendy A. , Park Yong Ho , Evans Jeremy N. , Kang ChulHee
Published: December 2004

The shikimate pathway synthesizes aromatic amino acids and other essential metabolites that are necessary for bacteria, plants and fungi to survive. This pathway is not present in vertebrates and therefore represents an attractive target for antibacterial agents. We have successfully crystallized and solved the structure of unliganded, inhibitor‐liganded and tetrahedral intermediate (TI)‐liganded forms of Streptococcus pneumoniae EPSP synthase. The overall topology of the S. pneumoniae EPSP synthase is similar to that of the Escherichia coli EPSP synthase. In addition, the majority of residues responsible for ligand binding were conserved between the two proteins. TI‐liganded structure provides absolute configuration of the C‐2 atom from the F‐PEP moiety of the enzyme‐bound intermediate and also defines key residues responsible for the enzyme reaction. Comparison of the unliganded state and substrate‐bound state of the enzyme provides insights into the structural mechanisms involved in dynamic events of ligand binding, domain movement and closure. This structural study of the pathogenic bacteria S. pneumoniae EPSP synthase with inhibitor and TI will provide invaluable information for the design of new‐generation antibiotics.
The Pseudomonas aeruginosa RpoS regulon and its relationship to quorum sensing
Authors: Schuster Martin , Hawkins Andrew C. , Harwood Caroline S. , Greenberg E. P.
Published: December 2004

In Escherichia coli and some other γ‐Proteobacteria, the alternative σ factor RpoS functions as a regulator of the general stress response. The role of RpoS in Pseudomonas aeruginosa is not clear. Although P. aeruginosa RpoS contributes to the resistance to several environmental stresses, its role appears to be less pivotal than in E. coli. In P. aeruginosa, RpoS also regulates the production of several virulence factors and influences the expression of individual genes that are controlled by quorum sensing. Some quorum‐controlled genes are induced by RpoS, whereas others are repressed. To gain insights about RpoS function in P. aeruginosa and to understand better the regulation of quorum‐controlled genes, we used transcript profiling to define an RpoS regulon. We identified 772 genes regulated by RpoS in stationary but not in logarithmic growth phase (504 were induced and 268 were repressed), and we identified putative RpoS promoter sequence elements with similarity to the E. coli RpoS consensus in several of these genes. Many genes in the regulon, for example a set of chemotaxis genes, have assigned functions that are distinct from those in E. coli and are not obviously related to a stress response. Furthermore, RpoS affects the expression of more than 40% of all quorum‐controlled genes identified in our previous transcriptome analysis. This highlights the significance of RpoS as a global factor that controls quorum‐sensing gene expression at the onset of stationary phase. The transcription profiling results have allowed us to build a model that accommodates previous seemingly conflicting reports.
Metabolic block at early stages of the glycolytic pathway activates the Rcs phosphorelay system via increased synthesis of dTDP‐glucose in Escherichia coli
Authors: El‐Kazzaz Waleed , Morita Teppei , Tagami Hideaki , Inada Toshifumi , Aiba Hiroji
Published: December 2004

A mutational block in the early stages of the glycolytic pathway facilitates the degradation of the ptsG mRNA encoding the major glucose transporter IICBGlc in Escherichia coli. The degradation is RNase E dependent and is correlated with the accumulation of either glucose‐6‐P or fructose‐6‐P (Kimata et al., 2001, EMBO J 20: 3587–3595; Morita et al., 2003, J Biol Chem 278: 15608–15614). In this paper, we investigate additional physiological effects resulting from the accumulation of glucose‐6‐P caused by a mutation in pgi encoding phosphoglucose isomerase, focusing on changes in gene expression. The addition of glucose to the pgi strain caused significant growth inhibition, in particular in the mlc background. Cell growth then gradually resumed as the level of IICBGlc decreased. We found that the transcription of the cps operon, encoding a series of proteins responsible for the synthesis of colanic acid, was markedly but transiently induced under this metabolic stress. Both genetic and biochemical studies revealed that the metabolic stress induces cps transcription by activating the RcsC/YojN/RcsB signal transduction system. Overexpression of glucose‐6‐P dehydrogenase eliminated both growth inhibition and cps induction by reducing the glucose‐6‐P level. Mutations in genes responsible for the synthesis of glucose‐1‐P and/or dTDP‐glucose eliminated the activation of the Rcs system by the metabolic stress. Taken together, we conclude that an increased synthesis of dTDP‐glucose activates the Rcs phosphorelay system, presumably by affecting the synthesis of oligosaccharides for enterobacterial common antigen and O‐antigen.
Topological analysis of Hin‐catalysed DNA recombination in vivo and in vitro
Authors: Merickel Stacy K. , Johnson Reid C.
Published: December 2004

In vitro
studies have demonstrated that Hin‐catalysed site‐specific DNA inversion occurs within a tripartite invertasome complex assembled at a branch on a supercoiled DNA molecule. Multiple DNA exchanges within a recombination complex (processive recombination) have been found to occur with particular substrates or reaction conditions. To investigate the mechanistic properties of the Hin recombination reaction
in vivo
, we have analysed the topology of recombination products generated by Hin catalysis in growing cells. Recombination between wild‐type recombination sites
in vivo
is primarily limited to one exchange. However, processive recombination leading to knotted DNA products is efficient on substrates containing recombination sites with non‐identical core nucleotides. Multiple exchanges are limited by a short DNA segment between the Fis‐bound enhancer and closest recombination site and by the strength of Fis–Hin interactions, implying that the enhancer normally remains associated with the recombining complex throughout a single exchange reaction, but that release of the enhancer leads to multiple exchanges. This work confirms salient mechanistic aspects of the reaction
in vivo
and provides strong evidence for the propensity of plectonemically branched DNA in prokaryotic cells. We also demonstrated that a single DNA exchange resulting in inversion
in vitro is accompanied by a loss of four negative supercoils.
Divergent binding sites on intercellular adhesion molecule‐1 (ICAM‐1) for variant Plasmodium falciparum isolates
Authors: Tse Man Tsuey , Chakrabarti Kausik , Gray Carolyn , Chitnis Chetan E. , Craig Alister
Published: December 2004

Adhesion of human erythrocytes infected with the malaria parasite Plasmodium falciparum to host endothelium has been associated with severe forms of this disease. A number of endothelial receptors have been identified, and there is evidence that one of these, intercellular adhesion molecule‐1 (ICAM‐1), may play an important role in the pathology of cerebral malaria. Mutagenesis of domain 1 of ICAM‐1, which is involved in parasite adhesion, shows that the binding sites for different parasite variants overlap to a large extent, but that there are subtle differences between them that correlate with their adhesive phenotypes. This suggests that the ability to bind to ICAM‐1 has arisen from a common variant, but that subsequent changes have led to differences in binding avidity, which may affect pathogenesis. The definition of common binding determinants and the elucidation of links between ICAM‐1 binding phenotype and disease will provide new leads in the design of therapeutic interventions.
Cellular engineering in a minimal microbe: structure and assembly of the terminal organelle of Mycoplasma pneumoniae
Authors: Krause Duncan C. , Balish Mitchell F.
Published: December 2004

Mycoplasma pneumoniae
is a minimal microbe with respect to cell envelope composition, biosynthetic and regulatory capabilities and genome size, yet it possesses a remarkably complex, multifunctional terminal organelle. This membrane‐bound extension of the mycoplasma cell is defined by the presence of an electron‐dense core that appears as paired, parallel bars oriented longitudinally and enlarging at the distal end to form a terminal button. Most non‐cytadhering mutants of
M. pneumoniae isolated to date exhibit defects in the architecture of the terminal organelle. Detailed characterization of those mutants has revealed the identities of many component proteins of the terminal organelle as well as the likely order in which some of those components are required. Additional questions regarding the composition of the electron‐dense core, the means by which the terminal organelle is duplicated during cell division and the manner in which this process is regulated remain to be answered. Thus, it seems that there is much to be learned about cellular engineering and spatial regulation in these ‘simple’ cell wall‐less bacteria.
Intein harbouring large tandem repeats in replicative DNA helicase of Trichodesmium erythraeum
Authors: Yang Jing , Meng Qing , Liu Xiang‐Qin
Published: December 2004

Protein splicing inteins can be small as ∼130 aa or up to ∼600 aa when harbouring an endonuclease domain. Here we report the identification and characterization of an unusually large intein, 1650 aa long and the largest of known inteins, encoded by the replicative DNA helicase gene dnaB of the oceanic N2‐fixing cyanobacterium Trichodesmium erythraeum. This Ter DnaB‐1 intein co‐exists with a 177‐aa mini‐intein in the same host protein and harbours large tandem repeats in which an 84‐aa sequence is repeated 16 times. Comparison between this tandem repeats and the recently reported tandem repeats of Ter DnaE‐1 intein revealed differences and similarities. The two tandem repeats, residing in different inteins of different host proteins, differ by 50% in size and have little sequence similarity. Tandem repeats in the Ter DnaB‐1 intein were required for the protein splicing activity when tested in Escherichia coli, in contrast to tandem repeats of the Ter DnaE‐1 intein that inhibited protein splicing. On the other hand, tandem repeats of both inteins are located in the same corresponding region of the intein sequence and have the same number of repeating units. These suggest that the two tandem repeats could be related but have diverged greatly in size, sequence and effect on protein splicing. Alternatively, they could have independent origins but evolved certain similarities because of common constraints in structure and maintenance.
Differences in two Pseudomonas aeruginosa cbb3 cytochrome oxidases
Authors: Comolli James C. , Donohue Timothy J.
Published: December 2004

Bacterial cytochrome cbb3 oxidases are members of the haeme‐copper oxidase superfamily that are important for energy conservation by a variety of proteobacteria under oxygen‐limiting conditions. The opportunistic pathogen Pseudomonas aeruginosa is unusual in possessing two operons that each potentially encode a cbb3 oxidase (cbb3‐1 or cbb3‐2). Our results demonstrate that, unlike typical enzymes of this class, the cbb3‐1 oxidase has an important metabolic function at high oxygen tensions. In highly aerated cultures, cbb3‐1 abundance and expression were greater than that of cbb3‐2, and only loss of cbb3‐1 influenced growth. Also, the activity of cbb3‐1, not cbb3‐2, inhibited expression of the alternative oxidase CioAB and thus influenced a signal transduction pathway much like that found in the α‐proteobacterium Rhodobacter sphaeroides. Cbb3‐2 appeared to play a more significant role under oxygen limitation by nature of its increased abundance and expression compared to highly aerated cultures, and the regulation of the cbb3‐2 operon by the putative iron‐sulphur protein Anr. These results indicate that each of the two P. aeruginosa cbb3 isoforms have assumed specialized energetic and regulatory roles.
Identification and characterization of NleA, a non‐LEE‐encoded type III translocated virulence factor of enterohaemorrhagic Escherichia coli O157:H7
Authors: Gruenheid Samantha , Sekirov Inna , Thomas Nikhil A. , Deng Wanyin , O'Donnell Paul , Goode David , Li Yuling , Frey Elizabeth A. , Brown Nathaniel F. , Metalnikov Pavel , Pawson Tony , Ashman Keith , Finlay B. Brett
Published: December 2004

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 uses a specialized protein translocation apparatus, the type III secretion system (TTSS), to deliver bacterial effector proteins into host cells. These effectors interfere with host cytoskeletal pathways and signalling cascades to facilitate bacterial survival and replication and promote disease. The genes encoding the TTSS and all known type III secreted effectors in EHEC are localized in a single pathogenicity island on the bacterial chromosome known as the locus for enterocyte effacement (LEE). In this study, we performed a proteomic analysis of proteins secreted by the LEE‐encoded TTSS of EHEC. In addition to known LEE‐encoded type III secreted proteins, such as EspA, EspB and Tir, a novel protein, NleA (non‐LEE‐encoded effector A), was identified. NleA is encoded in a prophage‐associated pathogenicity island within the EHEC genome, distinct from the LEE. The LEE‐encoded TTSS directs translocation of NleA into host cells, where it localizes to the Golgi apparatus. In a panel of strains examined by Southern blot and database analyses, nleA was found to be present in all other LEE‐containing pathogens examined, including enteropathogenic E. coli and Citrobacter rodentium, and was absent from non‐pathogenic strains of E. coli and non‐LEE‐containing pathogens. NleA was determined to play a key role in virulence of C. rodentium in a mouse infection model.
The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E
Authors: Boxma Brigitte , Voncken Frank , Jannink Sander , Van Alen Theo , Akhmanova Anna , Van Weelden Susanne W. H. , Van Hellemond Jaap J. , Ricard Guenola , Huynen Martijn , Tielens Aloysius G. M. , Hackstein Johannes H. P.
Published: December 2004

Anaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end‐products of a prokaryotic‐type mixed‐acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of pyruvate, which is in marked contrast to the hydrogenosomal metabolism of the anaerobic parabasalian flagellates Trichomonas vaginalis and Tritrichomonas foetus, because these organisms decarboxylate pyruvate with the aid of pyruvate:ferredoxin oxidoreductase (PFO). Here, we show that the chytrids Piromyces sp. E2 and Neocallimastix sp. L2 also possess an alcohol dehydrogenase E (ADHE) that makes them unique among hydrogenosome‐bearing anaerobes. We demonstrate that Piromyces sp. E2 routes the final steps of its carbohydrate catabolism via PFL and ADHE: in axenic culture under standard conditions and in the presence of 0.3% fructose, 35% of the carbohydrates were degraded in the cytosol to the end‐products ethanol, formate, lactate and succinate, whereas 65% were degraded via the hydrogenosomes to acetate and formate. These observations require a refinement of the previously published metabolic schemes. In particular, the importance of the hydrogenase in this type of hydrogenosome has to be revisited.
Identification of the sequences required for chromosomal replicator function in Kluyveromyces lactis
Authors: Irene Carmela , Maciariello Clelia , Cioci Francesco , Camilloni Giorgio , Newlon Carol S. , Fabiani Lucia
Published: December 2004

The analysis of replication intermediates of a Kluyveromyces lactis chromosomal autonomous replicating sequence (ARS), KARS101, has shown that it is active as a chromosomal replicator. KARS101 contains a 50 bp sequence conserved in two other K. lactis ARS elements. The deletion of the conserved sequence in KARS101 completely abolished replicator activity, in both the plasmids and the chromosome. Gel shift assays indicated that this sequence binds proteins present in K. lactis nuclear extracts, and a 40 bp sequence, previously defined as the core essential for K. lactis ARS function, is required for efficient binding. Reminiscent of the origin replication complex (ORC), the binding appears to be ATP dependent. A similar pattern of protection of the core was seen with in vitro footprinting. KARS101 also functions as an ARS sequence in Saccharomyces cerevisiae. A comparative study using S. cerevisiae nuclear extracts revealed that the sequence required for binding is a dodecanucleotide related to the S. cerevisiae ARS consensus sequence and essential for S. cerevisiae ARS activity.
Overproduction of the Lon protease triggers inhibition of translation in Escherichia coli: involvement of the yefM‐yoeB toxin‐antitoxin system
Authors: Christensen Susanne K. , Maenhaut‐Michel Geneviève , Mine Natacha , Gottesman Susan , Gerdes Kenn , Van Melderen Laurence
Published: December 2004

In Escherichia coli, the Lon ATP‐dependent protease is responsible for degradation of several regulatory proteins and for the elimination of abnormal proteins. Previous studies have shown that the overproduction of Lon is lethal. Here, we showed that Lon overproduction specifically inhibits translation through at least two different pathways. We have identified one of the pathways as being the chromosomal yefM‐yoeB toxin‐antitoxin system. The existence of a second pathway is demonstrated by the observation that the deletion of the yefM‐yoeB system did not completely suppress lethality and translation inhibition. We also showed that the YoeB toxin induces cleavage of translated mRNAs and that Lon overproduction specifically activates YoeB‐dependent mRNAs cleavage. Indeed, none of the other identified chromosomal toxin‐antitoxin systems (relBE, mazEF, chpB and dinJ‐yafQ) was involved in Lon‐dependent lethality, translation inhibition and mRNA cleavage even though the RelB and MazE antitoxins are known to be Lon substrates. Based on our results and other studies, translation inhibition appears to be the key element that triggers chromosomal toxin‐antitoxin systems. We propose that under Lon overproduction conditions, translation inhibition is mediated by Lon degradation of a component of the YoeB‐independent pathway, in turn activating the YoeB toxin by preventing synthesis of its unstable YefM antidote.
Large‐scale identification of essential Salmonella genes by trapping lethal insertions
Authors: Knuth Karin , Niesalla Heide , Hueck Christoph J. , Fuchs Thilo M.
Published: December 2004

A novel screening approach based on insertion‐duplication mutagenesis (IDM) was established to efficiently screen for essential genes of Salmonella enterica serovar Typhimurium under laboratory conditions. Small, randomly generated genomic fragments were cloned into a conditionally replicating vector, and the resulting library of single Salmonella clones was grown under permissive conditions. Upon switching to non‐permissive temperature, discrimination between lethal and non‐lethal insertions following homologous recombination allowed the trapping of genes with essential functions. Further characterization of a total of 498 fragments resulting in such lethal knockout revealed 145 known essential genes and 112 functionally characterized or hypothetical genes not yet shown to encode essential genes, among them three Salmonella‐specific genes. The essentiality was demonstrated for a prioritised set of 15 putative indispensable genes by creating conditional lethal phenotypes. The results of this large‐scale screening indicate that in rich media, the class of Salmonella genes indispensable for growth is composed of approximately 490 genes.