bluuebunny/crossref_metadata_2025 huggingface dataset:
A role for bacteriophages in the evolution and transfer of bacterial virulence determinants
Authors: Cheetham Brian F. , Katz Margaret E.
Published: March 2004

A virulence‐associated region in the genome of Dichelobacter nodosus has been shown to contain an integrase gene which is highly related to the integrases of Shigella flexneri phage Sf6 and coliphages P4 and φR73, together with open reading frames (vapB, C and D) related to genes borne on plasmids in Neisseria gonorrhoeae, Escherichia coli, Actinobacillus actinomycetemcomitans and Treponema denticola. Similar to P4 and φR73, the vap region is bracketed by putative bacteriophage att sites and is adjacent to a tRNA gene, which suggests that the vap region has been derived by the integration of a bacteriophage, or a plasmid carrying a bacteriophage‐related integrase gene. Many similarities in genes and genes clusters encoding virulence determinants have been found in distantly related bacteria. These genes are often located on plasmids in one organism but on the chromosome in others, implying that transmission of the genes has been followed by integration. Thus, the events which have generated the vap regions of D. nodosus may represent a common mechanism for transfer of virulence determinants. A number of genes involved in the virulence of bacterial pathogens are found on integrated bacteriophages, and we suggest that others will prove to be associated with tRNA genes and/or integrase genes derived from bacteriophages. The use of tRNA genes as integration sites for many bacteriophages and plasmids may favour intergeneric transmission, as tRNA genes are highly conserved.
Lipopolysaccharide with an altered O‐antigen produced in Escherichia coli K‐12 harbouring mutated, cloned Shigella flexneri rfb genes
Authors: Morona Renato , Macpherson Debbie F. , Van Den Bosch Luisa , Carlin Nils I.A. , Manning Paul A.
Published: March 2004

Cloning of the rfb genes of Shigella flexneri 2a into Escherichia coli K‐12 strain DH1 results in the synthesis of lipopolysaccharides (LPS) with an O‐antigen chain having type antigen IV and group antigens 3,4. During genetic studies of these rfb genes in E. coli K‐12, we observed that strains harbouring plasmids with certain mutations (inversion and transposon insertions) which should have blocked O‐antigen synthesis nevertheless still produced LPS with O‐antigen chains. These LPS migrated differently on silver‐stained SDS—polyacrylamide gels, compared with the LPS produced by wild‐type rfb genes, and the group 3,4 antigens were barely detectable, suggesting that the O‐antigen was altered. Investigation of the genetic determinants for production of the altered O‐antigen/LPS indicated that: (i) these LPS are produced as a result of mutations which are either polar on rfbF or inactivate rfbF; (ii) the rfbX gene product (or a similar protein in the E. coli K‐12 rfb region) is needed for production of the altered O‐antigen in the form of LPS; (iii) the rfbG gene product is required for the production of both the parental and altered LPS; (iv) the dTDP‐rhamnose biosynthesis genes are required. Additionally, an E. coli K‐12 gene product(s) encoded outside the rfb region also contributes to production of the O‐antigen of the altered LPS. An antiserum raised to the altered LPS from strain DH1(pPM2217 (rfbX::Tn1725)) was found to cross‐react with nearly all S. flexneri serotypes, and with the altered LPS produced by other DH1 strains harbouring plasmids with different rfb mutations, as described above. The reactivity of the altered LPS with a panel of monoclonal antibodies specific for various S. flexneri O‐antigen type and group antigens demonstrated that their O‐antigen components were closely related to that of S. flexneri serotype 4. The RfbF and RfbG proteins were shown to have similarity to rhamnose transferases, and we identified a motif common to the N‐termini of 6‐deoxy‐hexose nucleotide sugar transferases. We propose that the E. coli K‐12 strains harbouring the mutated S. flexneri rfb genes produce LPS with a hybrid O‐antigen as a consequence of inactivation of RfbF and complementation by an E. coli K‐12 gene product. Analysis of the genetic and immunochemical data suggested a possible structure for the O‐antigen component of the altered LPS.
Induction of complex intracytoplasmic membranes related to nitrogen fixation in Azoarcus sp. BH72
Authors: Hurek Thomas , Van Montagu Marc , Kellenberger Eduard , Reinhold‐Hurek Barbara
Published: March 2004

We report the discovery of novel subcellular structures related to bacterial nitrogen fixation in the strictly respiratory diazotrophic bacterium Azoarcus sp. BH72, which was isolated as an endophyte from Kallar grass. Nitrogenase is derepressed under microaerobic conditions at O2 concentrations in the micromolar range. With increasing O2 deprivation, bacteria can develop into a hyperinduced state, which is characterized by high specific rates of respiration and efficient nitrogen fixation at approximately 30 nM O2. Ultrastructural analysis of cells in the course of hyperinduction revealed that complex intracytoplasmic membrane systems are formed, which consist of stacks of membranes and which are absent under standard nitrogen‐fixing conditions. The iron protein of nitrogenase was highly enriched on these membranes, as evidenced by immunohistochemical studies. Membrane deficiency in NifH/K− mutants, a deletion mutant in the nifK gene and the character of NH4+‐grown cells suggested, in concert with the membrane localization of nitrogenase, that these structures are specialized membranes related to nitrogen fixation. We propose the term ‘diazosomes’ for them. Development of intracytoplasmic membranes coincides with the appearance of a high‐molecular‐mass form of the iron protein of nitrogenase, which was detectable in membrane fractions. Mutational analysis, and determination of the N‐terminal amino acid sequence indicate that the nifH gene product is covalently modified by a mechanism probably different from adenosine diphosphoribosylation. Development of diazosomes in nitrogen‐fixing cells can be induced in pure cultures and in co‐culture with a fungus isolated from the rhizosphere of Kallar grass.
ToIC and DsbA are needed for the secretion of STB, a heat‐stable enterotoxin of Escherichia coli
Authors: Foreman Devin T. , Martinez Ygnacio , Coombs Gary , Torres Alfredo , Kupersztoch Yankel M.
Published: March 2004

STB secretion‐deficient mutants were isolated using the synthetic transposon TnβIaM. Cultures were plated using a double‐membrane system of cellulose acetate and nitrocellulose placed on Luria agar plates containing carbenicillin. The STB bound to the underlying nitrocellulose membrane was detected with anti‐STB antibodies. The altered genes of two STB secretion‐deficient mutants were identified by conjugation and complementation as toIC and dsbA. In cultures of well‐characterized dsbA and toIC mutants, STB was absent from the culture supernatant. The role of ToIC and DsbA in the secretion of peptides is discussed.
Regulation of the heat‐shock response depends on divalent metal ions in an hfIB mutant of Escherichia coli
Authors: Herman Christophe , Lecat Sandra , D'Ari Richard , Bouloc Philippe
Published: March 2004

HfIB, also called FtsH, is an essential Escherichia coli protein involved in the proteolysis of the heat‐shock regulator σ32 and of the phage regulator λcll. The hfIB1(Ts) allele (formerly called ftsH1) conferring temperature‐sensitive growth at 42°C is suppressed by loss of the ferric‐uptake repressor Fur and by anaerobic growth. We show here that suppression requires TonB‐dependent Fe(III) transport in the hfIB1(Ts) fur mutant during aerobic growth at 42°C and Feo‐dependent Fe(II) transport during anaerobic growth at 42°C. Temperature‐resistant growth of hfIB1(Ts) strains is also observed at 42°C in the presence of a high concentration of Fe(II), Ni(II), Mn(II) or Co(II) salts, but not in the presence of Zn(II), Cd(II), Cu(II), Mg(II), Ca(II) or Cr(III) salts. However, neither Ni(II) nor a fur mutation permits growth in the complete absence of HfIB. The heat‐shock response, evaluated by an htpG::lacZ fusion, is overinduced in hfIB1(Ts) strains at 42°C because of stabilization of σ32. Growth in the presence of Ni(II) or in the absence of the Fur repressor abolishes this overinduction in the hfIB1(Ts) strain, and, in the hfIB1(Ts) fur mutant, σ32 is no longer stabilized at 42°C. These results reinforce the recent observation that HfIB is a metalloprotease active against σ32in vitro and suggest that it can associate functionally in vivo with Fe(II), Ni(II), Mn(II) and Co(II) ions.
Evidence for lateral transfer and recombination in OspC variation in Lyme disease Borrelia
Authors: Livey I. , Gibbs C.P. , Schuster R. , Dorner F.
Published: March 2004

The ospC gene was amplified by the polymerase chain reaction from each of 76 Lyme disease Borrelia strains. Restriction fragment length polymorphism (RFLP) analysis demonstrated 33 distinct RFLP types; two additional RFLP types were identified from published ospC sequences. For each RFLP type, at least one ospC gene was sequenced and the degree of sequence relatedness examined by construction of an ospC gene tree. The genes were extremely diverse, with sequence identity ranging from 74.4% to 99.0%; the majority of changes are localized within the central portion of the molecule. A comparison of ospC sequences suggests that recombination occurs frequently between ospC alleles; this genetic exchange is proposed to be mediated by lateral transfer of ospC sequences. Evidence indicates that recombination occurs between ospC genes from the same Borrelia species (i.e. B. afzelii and B. garinii) as well as between different Borrelia species (i.e. B. afzelii and B. garinii, B. burgdorferi and genogroup DN127).
pAMβ1 resolvase has an atypical recombination site and requires a histone‐like protein HU
Authors: Petit Marie‐Agnès , Ehrlich Dusko , Jannière Laurent
Published: March 2004

The broad‐host‐range plasmid pAMβ1 from Gram‐positive bacteria encodes a resolvase, designated Resβ, which shares homology with the proteins of the resolvase—invertase family. Here we report the purification and in vitro characterization of Resβ. This resolvase is particular in two aspects: it has an atypical binding site and requires a cofactor to promote resolution in vitro. Resβ binds to two regions within its resolution site res. One contains two inverted repeats (R1 and R2), the other contains only one repeat (R3). The cofactor required for resolution in vitro is present in crude extracts of both Bacillus subtilis and Escherichia coli and can be substituted by the E. coli histone‐like protein HU. The possible mode of action of HU in the resolution process is discussed.
Identification of mycoplasma membrane proteins by systematic TnphoA mutagenesis of a recombinant library
Authors: Cleavinger Catherine M. , Kim Mary F. , Im Jeong H. , Wise Kim S.
Published: March 2004

Wall‐less prokaryotes in the genus Mycoplasma include over 90 species of infectious agents whose pathogenicity for humans and other animals is currently being assessed. Molecular characterization of surface proteins is critical in this regard but is hampered by the lack of genetic systems in these organisms. We used TnphoA transposition to systematically mutagenize, in Escherichia coli, a genomic plasmid library constructed from Mycoplasma fermentans, a potential human pathogen. The strategy circumvented problems of expressing mycoplasma genes containing UGA (Trp) codons and relied on the construction of the vector pG7ZCW, designed to reduce TnphoA transposition into vector sequences. Functional phoA gene fusions directly identified genes encoding 19 putative membrane‐associated proteins of M. fermentans. Sequences of fusion constructs defined three types of export sequence: (1) non‐cleavable, membrane‐spanning sequences, (2) signal peptides with signal peptidase (SPase) I‐like cleavage sites, and (3) signal peptides with SPase II‐like lipoprotein‐cleavage sites which, like most other mycoplasmal lipoprotein signals analysed to date, differed from those in several Gram‐negative and Gram‐positive eubacteria in their lack of a Leu residue at the −3 position. Antibodies to synthetic peptides that were deduced from two fusions to predicted lipoproteins, identified corresponding amphiphilic membrane proteins of 57 kDa and 78 kDa expressed in the mycoplasma. The P57 sequence contained a proline‐rich N‐terminal region analogous to an adhesin of Mycoplasma gallisepticum. The P78 protein was identical to a serologically defined phase‐variant surface lipoprotein. TnphoA mutagenesis provides an efficient means of systematically characterizing functionally diverse lipoproteins and other exported proteins in mycoplasmas.
Bacillus subtilis MrgA is a Dps(PexB) homologue: evidence for metalloregulation of an oxidative‐stress gene
Authors: Chen Lei , Helmann John D.
Published: March 2004

Upon the cessation of exponential growth, Bacillus subtilis enters a transition phase leading to either sporulation or a non‐sporulating stationary phase. During this transition period, cells secrete degradative enzymes, become competent for DNA transformation, are motile and acquire resistance to oxidative killing. We now report that mrgA, originally identified as a gene repressed by metal ions, encodes a member of the Dps/PexB family of general stress proteins. Like Escherichia coli Dps(PexB), MrgA forms highly stable, multimeric protein‐DNA complexes which accumulate in stationary‐phase cells and protect against oxidative killing. MrgA is part of an inducible oxidative stress response in B. subtilis: mrgA is induced by hydrogen peroxide, and a strain lacking MrgA displays increased sensitivity to oxidative killing. In addition, a hydrogen peroxide‐resistant mutant, which constitutively overproduces catalase and alkyl hydroperoxide reductase, also overproduces MrgA. These results indicate a complex interplay between metal ions and the expression of the B. subtilis oxidative stress response.
sbmC, a stationary‐phase induced SOS Escherichia coli gene, whose product protects cells from the DNA replication inhibitor microcin B17
Authors: Baquero M.R. , Bouzon M. , Varea J. , Moreno F.
Published: March 2004

Microcin B17 (MccB17) is a ribosomally synthesized peptide antibiotic of 43 amino acids that induces double‐strand breaking of DNA in a DNA gyrase‐dependent reaction. As a consequence, the SOS regulon is induced and massive DNA degradation occurs. In this work we have characterized an Escherichia coli gene, sbmC, that in high copy number determines high cell resistance to MccB17. sbmC encodes a cytoplasmic polypeptide of 157 amino acids (Mr, 18 095) that has been visualized in SDS—polyacrylamide gels. The gene is located at min 44 of the E. coli genetic map, close to the sbcB gene. sbmC expression is induced by DNA‐damaging agents and, also, by the entry of cells into the stationary growth phase. A G → T transversion at the fifth nucleotide of the quasicanonical LexA‐box preceding the gene makes recA cells 16‐fold more resistant to exogenous MccB17. The gene product, SbmC, also blocks MccB17 export from producing cells. Altogether, our results suggest that SbmC recognizes and sequesters MccB17 in a reversible way.
Characterization of an Escherichia coli rotA mutant, affected in periplasmic peptidyl‐prolyl cis/trans isomerase
Authors: Kleerebezem Michiel , Heutink Marja , Tommassen Jan
Published: March 2004

The rotA gene of Escherichia coli encodes a peptidyl‐prolyl cis/trans isomerase (PPlase), which is supposed to catalyse protein folding in the periplasm. To investigate the importance of the enzyme, the rotA gene was cloned and a chromosomal deletion mutant was created. The rotA mutant was normally viable. No residual PPlase activity could be detected in the periplasmic fraction of the mutant. Comparison of the patterns of periplasmic and outer membrane proteins by SDS‐PAGE revealed no differences in protein composition between the rotA mutant and its parental strain. Similarly, the kinetics of periplasmic protein folding and outer membrane protein assembly appeared unaffected by the rotA mutation. Our results show that the periplasmic PPlase of E. coli is not essential and that the protein does not play an important role in protein folding.
Deletion analysis of gene minE which encodes the topological specificity factor of cell division in Escherichia coli
Authors: Pichoff Sébastien , Vollrath Benedikt , Touriol Christian , Bouché Jean‐Pierre
Published: March 2004

Division inhibition caused by the minCD gene products of Escherichia coli is suppressed specifically at mid‐cell by MinE protein expressed at physiological levels. Excess MinE allows division to take place also at the poles, leading to a minicell‐forming (Min−) phenotype. In order to investigate the basis of this topological specificity, we have analysed the ability of truncated derivatives of MinE to suppress either minCD‐dependent division inhibition in a chromosomal Δ(minB) background, or the division inhibition exerted by MinCD at the cell poles in a minB,+ strain. Our results indicate that these two effects are not mediated by identical interactions of MinE protein. In addition, gel filtration and the yeast two‐hybrid system indicated that MinE interacts with itself by means of its central segment. Taken together, our results favour a model in which wild‐type MinE dimer molecules direct the division inhibitor molecules to the cell poles, thus preventing polar divisions and allowing non‐polar sites to divide. This model explains how excess MinE, or an excess of certain MinE derivatives which prevent the accumulation of the division inhibitor at the poles, can confer a Min− phenotype in a minB+ strain.
Fnr activates transcription from the P6 promoter of the pfl operon in vitro
Authors: Kaiser Manuela , Sawers Gary
Published: March 2004

Expression of the Escherichia coli focA—pfl operon is induced by anaerobiosis and transcription is controlled by seven promoters. Anaerobic induction is mediated by the ArcA and Fnr transcription factors. Fnr was purified and its specific interaction with the pfl promoter‐regulatory region was characterized. A single binding site could be identified by DNase I footprinting, which was centred at −40.5 bp relative to the start site of promoter 6 (P6) transcription. Activation of P6 transcription in vitro was completely dependent on the Fnr protein. Fnr‐dependent transcription could be prevented by mutating the Fnr‐binding site, indicating that Fnr must bind to its recognition sequence to be able to activate transcription. An Fnr‐independent promoter, which overlaps the P6 promoter, was also identified and characterized in vivo and in vitro. Transcription from this promoter (termed P6A) initiated 10 bp upstream of the P6 start site and assures that low‐level synthesis of the FocA protein occurs under aerobic growth conditions.
Mutational analysis of the Yersinia enterocolitica virC operon: characterization of yscE, F, G, I, J, K required for Yop secretion and yscH encoding YopR
Authors: Allaoui Abdelmounaaïm , Schulte Ralf , Cornelis Guy R.
Published: March 2004

Pathogenic yersiniae secrete the Yop anti‐host proteins using a type‐III secretion pathway. The components of the secretion machinery are encoded by three loci on the pYV plasmid: virA, virB, and virC. In this paper we describe the characterization of eight non‐polar mutants of the virC locus, constructed by allelic exchange. The yscE, FG, I, J and K mutants were defective in Yop secretion and independent of Ca2+ (Cl) for their growth at 37°C. Substitution of the 12 N‐terminal amino‐acid residues of YscF impaired secretion of YopB and YopD only and led also to a Cl phenotype. The culture supernatant of the yscH mutant contained all the Yops except the 18 kDa YopR. Complementation experiments and an immunoblot analysis confirmed that YopR is encoded by the yscH gene. The LD50 for the mouse of the yscH mutant was 10‐fold higher than that of the parental strain indicating that YopR is involved in pathogenesis. The phenotype of the yscM mutant was similar to that of the wild‐type strain. However, overproduction of YscM from a multicopy plasmid in wild‐type Yersinia enterocolitica prevented Yop secretion and synthesis. A hybrid YopH—LacZ′ protein, encoded by a gene transcribed from the lac promoter, was secreted by a strain overexpressing YscM, showing that the secretion machinery was still functional. These results indicate that YscM plays a role in the feedback inhibition of Yop synthesis when secretion is compromised by acting as a negative regulator of Yop synthesis.
Distinct and differently regulated Mo‐dependent nitrogen‐fixing systems evolved for heterocysts and vegetative cells of Anabaena variabilis ATCC 29413: characterization of the fdxH1/2 gene regions as part of the nif1/2 gene clusters
Authors: Schrautemeier Bernhard , Neveling Ute , Schmitz Stefan
Published: March 2004

Two different fdxH genes (fdxH1, fdxH2) have been isolated from the nitrogen‐fixing, heterocyst‐forming cyanobacterium Anabaena variabilis ATCC 29413. They are part of two different nif gene clusters, nif1 and nif2. fdxH1 encodes the [2Fe—2S] ferredoxin that is known as the direct electron donor to nitrogenase in heterocysts, and is very similar to FdxH from Anabaena sp. PCC 7120. FdxH2 has more residues in common and shares its oxygen sensitivity with the single FdxH from the non‐heterocystous, filamentous cyanobacterium Plectonema boryanum PCC 73110. The latter expresses nitrogenase early ( 3–4 h) after nitrogen depletion in vegetative cells and exclusively under anaerobic conditions. fdxH2 and the nif2 genes of Anabaena 29413 are also transcribed 4 h after onset of nitrogen‐stepdown, exclusively under anaerobic growth conditions and long before functional heterocysts appear. At this time, no fdxH1 and nif1 gene transcription was observed. It occurred later and was associated with nitrogen fixation under aerobic conditions, i.e. within heterocysts. fdxH2 and nifHDK2 were not transcribed during aerobic, nitrogen‐fixing growth. In addition, neither was an fdxH2‐type gene found nor an anaerobically and early inducible Nif2 system detectable in Anabaena 7120. These data reveal that in filamentous cyanobacteria two different Nif systems have evolved based on molybdenum nitrogenases. It is concluded that a Nif2‐type system operates in vegetative cells of non‐heterocystous and some, but not all, heterocyst‐forming filamentous cyanobacteria. It is environmentally regulated by the levels of both oxygen and combined nitrogen in the habitat. To simultaneously allow for oxygen‐evolving photosynthesis and oxygen‐sensitive nitrogen fixation, the Nif1‐type system probably branched from an ancestral Nif2‐type system and has evolved for an exclusive operation within heterocysts. Accordingly, its expression has become an obligate late event in the developmental programme of heterocyst differentiation, irrespective of aerobic or anaerobic growth conditions.
Quelling the red menace: haem capture by bacteria
Authors: Lee B. Craig
Published: March 2004

Haem is an important bacterial nutrient. As a prosthetic group of several proteins, haem functions as a cofactor mediating oxygen transport, energy generation, and mixed‐function oxidation. In addition, the iron chelated in the porphyrin ring may serve as an iron substrate for growth. However, because of its propensity for oxidizing cellular constituents, haem is always associated with proteins. Therefore, the uptake and transit of haem across bacterial membranes requires the participation of protein escorts. Bacteria have evolved a diverse array of surface‐exposed receptors dedicated to binding haem and haem‐proteins. Following this selective recognition at the bacterial cell surface, haem is transported across the outer membrane via a TonB‐dependent process. The control of receptor expression appears to be multifactorial, probably involving a number of global regulators. A model integrating this information is presented.
Molecular cloning, sequence analysis, and functional characterization of the lipopolysaccharide biosynthetic gene kdtA encoding 3‐deoxy‐α‐d‐manno‐octulosonic acid transf erase of Chlamydia pneumoniae strain TW‐183
Authors: Löbau Sabine , Mamat Uwe , Brabetz Werner , Brade Helmut
Published: March 2004

The gene kdtA of Chlamydia pneumoniae strain TW‐183, encoding the enzyme 3‐deoxy‐α‐d‐manno‐octulosonic acid (Kdo)transferase of lipopolysaccharide biosynthesis, was cloned and sequenced. A single open reading frame of 1314 bp was identified, the deduced amino acid sequence of which revealed 69% similarity and 43% identity with KdtA of Chlamydia trachomatis and Chlamydia psittaci. The gene was expressed in the Gram‐positive host Corynebacterium glutamicum and the primary gene product was characterized as a multi‐functional glycosyltransferase. Cell‐free extracts generated in vitro the genus‐specific epitope of Chlamydia composed of the trisaccharide (αKdo(2–8)αKdo(2–4)αKdo. The results show that a single polypeptide affords three different glycosidic bonds, which is in contradiction to the dogma of glycobiology: ‘one enzyme — one glycosidic bond’.
Membrane glycerophospholipid biosynthesis in Neisseria meningitidis and Neisseria gonorrhoeae: identification, characterization, and mutagenesis of a lysophosphatidic acid acyltransferase
Authors: Swartley John S. , Balthazar Jacqueline T. , Coleman Jack , Shafer William M. , Stephens David S.
Published: March 2004

Lysophosphatidic acid (LPA) acyltransferases of Neisseria meningitidis and Neisseria gonorrhoeae were identified which share homology with other prokaryotic and eukaryotic LPA acyltransferases. In Escherichia coli, the conversion of LPA to phosphatidic acid, performed by the 1‐acyl‐sn‐glycerol‐3‐phosphate acyltransferase PlsC, is a critical intermediate step in the biosynthesis of membrane glycerophospholipids. A Tn916‐generated mutant of a serogroup B meningococcal strain was identified that exhibited increased amounts of capsular polysaccharide, as shown by colony immunoblots, and a threefold increase in the number of assembled pili. The single, truncated 3.8 kb Tn916 insertion in the meningococcal mutant was localized within a 771 bp open reading frame. The gonococcal equivalent of this gene was identified by transformation with the cloned meningococcal mutant gene. In N. gonorrhoeae, the mutation increased piliation fivefold. The insertions were found to be within a gene that was subsequently designated nIaA (neisserial LPA acyltransferase). The predicted neisserial LPA acyltransferases were homologous (>20% identity,>40% amino acid similarity) to the family of PlsC protein homologues. A cloned copy of the meningococcal nIaA gene complemented in trans a temperature‐sensitive E. coli PlsCts− mutant. Tn916 and Ω‐cassette insertional inactivations of the neisserial nIaA genes altered the membrane glycerophospholipid compositions of both N. meningitidis and N. gonorrhoeae but were not lethal. Therefore, the pathogenic Neisseria spp. appear to be able to utilize alternative enzyme(s) to produce phosphatidic acid. This hypothesis is supported by the observation that, although the amounts of mature glycerophospholipids were altered in the meningococcal and the gonococcal nIaA mutants, glycerophospholipid synthesis was detectable at significant levels. In addition, acyltransferase enzymatic activity, while reduced in the gonococcal nIaA mutant, was increased in the meningococcal nIaA mutant. We postulate that the pathogenic Neisseria spp. are able to utilize alternate acyltransferases to produce glycerophospholipids in the absence of nIaA enzymatic activity.Implementation of these secondary enzymes results in alterations of glycerophospholipid composition that lead to pleiotropic effects on the cell surface components, including effects on capsule and piliation.
The unrelated surface proteins ActA of Listeria monocytogenes and lcsA of Shigella flexneri are sufficient to confer actin‐based motility on Listeria innocua and Escherichia coli respectively
Authors: Kocks C. , Marchand J.‐B. , Gouin E. , D'Hauteville H. , Sansonetti P.J. , Carlier M.‐F. , Cossart P.
Published: March 2004

Listeria monocytogenes and Shigella flexneri are two unrelated facultative intracellular pathogens which spread from cell to cell by using a similar mode of intracellular movement based on continuous actin assembly at one pole of the bacterium. This process requires the asymmetrical expression of the ActA surface protein in L. monocytogenes and the lcsA (VirG) surface protein in S. flexneri. ActA and lcsA share no sequence homology. To assess the role of the two proteins in the generation of actin‐based movement, we expressed them in the genetic context of two non‐actin polymerizing, non‐pathogenic bacterial species, Listeria innocua and Escherichia coli. In the absence of any additional bacterial pathogenicity determinants, both proteins induced actin assembly and propulsion of the bacteria in cytoplasmic extracts from Xenopus eggs, as visualized by the formation of characteristic actin comet tails. E. coli expressing lcsA moved about two times faster than Listeria and displayed longer actin tails. However, actin dynamics (actin filament distribution and filament half‐lives) were similar in lcsA‐ and ActA‐induced actin tails suggesting that by using unrelated surface molecules, L. monocytogenes and S. flexneri move intracellularly by interacting with the same host cytoskeleton components or by interfering with the same host cell signal transduction pathway.
The amino‐terminal part of ActA is critical for the actin‐based motility of Listeria monocytogenes; the central proline‐rich region acts as a stimulator
Authors: Lasa Iñigo , David Violaine , Gouin Edith , Marchand Jean‐Baptiste , Cossar Pascale
Published: March 2004

The intracellular bacterial pathogen Listeria monocytogenes moves inside the host‐cell cytoplasm propelled by continuous actin assembly at one pole of the bacterium. This process requires expression of the bacterial surface protein ActA. Recently, in order to identify the regions of ActA which are required for actin assembly, we and others have expressed different domains of ActA by transfection in eukaryotic cells. As this type of approach cannot address the role of ActA in the actin‐driven bacterial propulsion, we have now generated several L. monocytogenes strains expressing different domains of ActA and analysed the ability of the different domains to trigger actin assembly and bacterial movement in both infected cells and cytoplasmic extracts. We show here that the amino‐terminal part is critical for F‐actin assembly and movement. The internal proline‐rich repeats and the carboxy‐terminal domains are not essential. However, in vitro motility assays have demonstrated that mutants lacking the proline‐rich repeats domain of ActA moved two times slower (6±2 µm min−1) than the wild type (13±3µm min−1}). In addition, phosphatase treatment of protein extracts of cells infected with the L. monocytogenes strains expressing the ActA variants suggested that phosphorylation may not be essential for ActA activity.
Deletion mutagenesis of Tn10 Tet repressor — localization of regions important for dimerization and inducibility in vivo
Authors: Berens Christian , Pfleiderer Klaus , Helbi Vera , Hillen Wolfgang
Published: March 2004

The gene for the Tn10 Tet repressor (TetR) was subjected to deletion mutagenesis. Screening for a transdominant operator‐binding negative phenotype yielded 10 mutants with internal deletions. Three deletions extend from residue D5 to residues L41, W75, or Q76, respectively, and two contain deletions of the α‐helix‐turn‐α‐helix DNA‐binding motif. Five deletions range from residue K84 to residues between R87 and K98. Since residues from the N‐terminus up to position 98 are not necessary for dimerization, this must take place in the C‐terminal half of the protein. Ability to dimerize was probed by introducing ochre non‐sense codons (oc) at residues G138, H151, E159, l174, or K202. Koc202 shows wild‐type in vivo operator‐binding and inducibility by tetracycline indicating that the six C‐terminal residues of TetR are not important for activity. Mutants with longer C‐terminal truncations are inactive and not transdominant. They show reduced steady‐state protein levels and are probably impaired in folding and degraded in vivo. Two mutants (Δ151–166, Δ164–166) with deletions in a region variable in primary structure and length among Tet repressers from different resistance determinants bind tet operator efficiently, but are not inducibie by tetracycline. This result indicates that these residues are not important for dimer formation in the operator‐binding form.
The Flp recombinase cleaves Holliday junctions in trans
Authors: Dixon Julie E. , Shaikh Arkady C. , Sadowski Paul D.
Published: March 2004

The Flp site‐specific recombinase is encoded by the 2 µm plasmid of Saccharomyces cerevisiae and is a member of the integrase family of recombinases. Like all members of the integrase family studied, Flp mediates recombination in two steps. First, a pair of strand exchanges creates a Holliday‐like intermediate; second, this intermediate is resolved to recombinant products by a second pair of strand exchanges.

Evidence derived from experiments using linear substrates indicates that Flp's active site is composed of two Flp protomers. One binds to the Flp recognition target site (FRT site) and activates the scissile phosphodiester bond for cleavage. Another molecule of Flp bound elsewhere in the synaptic complex (in trans) donates the nucleophilic tyrosine that executes cleavage and thereby becomes covalently attached to the 3′ phosphoryl group at the cleavage site.

It has previously been shown that Flp efficiently resolves synthetic, Holliday‐like (χ) structures to linear products. In this paper, we examined whether resolution of χ structures by Flp also occurs via the trans cleavage mechanism. We used in vitro complementation studies of mutant Flp proteins as well as nicked χ structures to show that Flp resolves χ structures by trans cleavage. We propose a model for Flp‐mediated recombination that incorporates trans cleavage at both the initial and resolution steps of strand exchange.
Visualization of the subcellular location of sporulation proteins in Bacillus subtilis using immunofluorescence microscopy
Authors: Pogliano Kit , Harry Elizabeth , Losick Richard
Published: March 2004

We describe the application of immunofluorescence microscopy to visualization of the subcellular localization of proteins involved in coat morphogenesis and chromosome packaging during the process of sporulation in Bacillus subtilis. In confirmation and extension of previous findings, we show that SpolVA, which is responsible for guiding coat formation to the surface of the outer membrane that surrounds the developing spore, assembles into a shell that is located close to or on the surface of this enveloping membrane. CotE, which is responsible for the formation of the outer layer of the coat, assembles into a second shell of apparently larger diameter. Assembly of SpolVA could be detected as early as the morphological stage of polar septation and closely followed the enveloping membrane of the mother cell during the stage of engulfment, thereby providing a sensitive and diagnostic marker for this phagocytic‐like process. Surprisingly, the chromosome of the developing spore and the small, acid‐soluble proteins, known as α/β‐type SASPs, that are known to coat the spore chromosome, were found to co‐localize to a doughnut‐like ring of approximately 1 µm in diameter. The use of a double mutant lacking the α/β‐type SASP demonstrated that these high abundance, DNA‐binding proteins are responsible for packaging the chromosome of the developing spore into this unusual structure. We conclude that sporulation in B. subtilis is a fertile system for addressing cell biological problems in a bacterium and that immunofluorescence microscopy provides a sensitive method for visualizing protein subcellular localization at high resolution.
The role of the chromatin‐associated protein Hbsu in β‐mediated DNA recombination is to facilitate the joining of distant recombination sites
Authors: Alonso Juan C. , Gutierrez Crisanto , Rojo Fernando
Published: March 2004

The β recombinase is unable to mediate in vitro DNA recombination between two directly oriented recombination sites unless a bacterial chromatin‐associated protein (Bacillus subtilis Hbsu or Eschrichia coli HU) is provided. By electron microscopy, we show that the role of Hbsu is to help in joining the recombination sites to form a stable synaptic complex. Some evidence supports the fact that Hbsu works by recognizing and stabilizing a DNA structure at the recombination site, rather than by serving as a bridge between β recombinase dimers through a protein‐protein interaction. We show that the mammalian HMG1 protein, which shares neither sequence nor structural homology with Hbsu, can also stimulate β‐mediated recombination. These chromatin‐associated proteins share the property of binding to DNA in a relatively non‐specific fashion, bending it, and having a marked preference for altered DNA structures. Hbsu, HU or HMG1 proteins probably bind specifically at the crossing‐over region, since at limiting protein‐DNA molar ratios they could not be outcompeted by an excess of a DNA lacking the crossing over site. Distamycin, a minor groove binder that induces local distortions in DNA, did not affect the binding of β protein to DNA, but inhibited the formation of the synaptic complex.
Salmonella typhimurium secreted invasion determinants are homologous to Shigella lpa proteins
Authors: Hueck Christoph J. , Hantman Michael J. , Bajaj Vivek , Johnston Christine , Lee Catherine A. , Miller Samuel I.
Published: March 2004

Salmonella typhimurium secreted proteins (Ssp) were previously implicated in epithelial cell invasion. Here we describe four genes (sspB, sspC, sspD, and sspA), located between spaT and prgH, which encode proteins of 63, 42, 36, and 87 kDa, respectively. These Ssp are homologous to Shigella flexneri secreted proteins lpaB, lpaC, lpaD and lpaA. A non‐invasive mutant with a transposon insertion in sspC lacks Ssp of 87,42 and 36 kDa. Complementation analyses show that sspC and sspD encode the 42 and the 36 kDa Ssp, while the 87 kDa Ssp is encoded by sspA. sspC and sspD, but not sspA are required for invasion. Amino‐terminal sequencing shows that SspC and SspA are secreted without amino‐terminal processing. We further demonstrate that Ssp secretion requires proteins encoded by prgHIJK, homologous to the Shigella lpa secretion system, since SspA is abundantly secreted by wild‐type bacteria but is completely retained within the cellular fraction of a prgHIJK mutant. A precipitate containing abundant SspC and three other major Ssp of 63,59 and 22 kDa was isolated from culture supernatants of wild‐type bacteria. These data indicate that major secreted invasion determinants of S. typhimurium are structurally and functionally homolgous to S. flexneri lpa proteins.
Mutational activation of the Cpx signal transduction pathway of Escherichia coli suppresses the toxicity conferred by certain envelope‐associated stresses
Authors: Cosma Christine L. , Danese Paul N. , Carlson John H. , Silhavy Thomas J. , Snyder William B.
Published: March 2004

The processing‐defective outer membrane porin protein LamBA23D (Carlson and Silhavy, 1993) and a tripartite fusion protein, LamB‐LacZ‐PhoA (Snyder and Silhavy, 1995), are both secreted across the cytoplasmic membrane of Escherichia coli, where they exert an extracytoplasmic toxicity. Suppressors of these toxicities map to a previously characterized gene, cpxA, that encodes the sensor kinase protein of a two‐component regulatory system. These activated cpxA alleles, designated as cpxA*, stimulate transcription of the periplasmic protease DegP (Danese et aL, 1995), which in turn catalyses degradation of the tripartite fusion protein. In contrast, degradation of precursor LamBA23D is not significantly stimulated in a cpxA* suppressor background. In fact, increased levels of DegP in a wild‐type background stabilized this protein. While a functional degP gene is required for full cpxaA* ‐mediated suppression of both toxic envelope proteins, residual suppression is seen in cpxA*degP::Tn10 double mutants. Furthermore, cpxA* mutations suppress the toxicity conferred by the LamB‐LacZ hybrid protein, which exerts its effects in the cytoplasm, sequestered from DegP. Together, these observations suggest that the activated Cpx pathway regulates additional downstream targets that contribute to suppression. A subset of these targets may constitute a regulon involved in relieving extracytoplasmic and/or secretion‐related stress.
Evidence for in vivo but not in vitro expression of a Borrelia burgdorferi outer surface protein F (OspF) homologue
Authors: Akins Darrin R. , Porcella Stephen F. , Popova Taissia G. , Shevchenko Dmitriy , Baker Scott I. , Li Minyue , Norgard Michael V. , Radolf Justin D.
Published: March 2004

Protein export signals from the low‐passage 297 strain of Borrelia burgdorferi were cloned as fusions with an Escherichia coli alkaline phosphatase (PhoA) reporter lacking a signal sequence. One PhoA+ clone (BbK2.10‐PhoA) was derived from a borrelial lipoprotein. Although the polypeptide encoded by the full‐length bbk2.10 gene had 76% similarity and 56% identity to outer surface protein F (OspF) from B. burgdoferi strain N40, antibodies directed against recombinant forms of the two proteins revealed that they were not cross‐reactive. The nucleotide sequences of bbk2.10 and ospF from the N40 and 297 strains, respectively, were determined to confirm that the N40 and 297 strains each contained both genes. Southern blot analysis revealed that bbk2.10 is a single‐copy gene and that the B. burgdorferi strain 297 and N40 genomes appeared to contain one other gene more closely related to ospF than bbk2.10. It was particularly note worthy that ospF, but not bbk2.10, was expressed in vitro while B. burgdorferi‐infected mice generated antibodies reactive with both lipoproteins. To help confirm that the BbK2.1O‐reactive antibodies produced by the B. burgdorferi‐infected mice were specific for that protein, a second gene, bbk2.11, which hybridized with the ospF probe was cloned; the corresponding polypeptide reacted strongly with OspF antisera but failed to react with BbK2.10‐specific antisera. Taken together, these data demon‐strate that BbK2.10, BbK2.11, and OspF comprise a B. burgdorferi lipoprotein family and that at least one member (BbK2.10) appears to be expressed only during infection.
Transcriptional co‐activation at the ansB promoters: involvement of the activating regions of CRP and FNR when bound in tandem
Authors: Scott Shaun , Busby Stephen , Beacham Ifor
Published: March 2004

Previous work with semi‐synthetic promoters containing a single CRP binding site centred at 41.5 bp from the transcription start site has demonstrated enhanced transcription (synergism) when a second binding site, for CRP or FNR, is placed upstream at around −91 bp. The ansB promoter in Escherichia coli is co‐activated in a co‐dependent manner by one dimer each of CRP and FNR protein whose binding sites are at around −91 and −41 bp, respectively, from the transcription start site. Similarly, the homologous ansB promoter in Salmonella is co‐activated by two dimers of CRP which function synergistically. The binding sites at the E. coli promoter have been changed by mutation to provide a number of active promoter derivatives carrying other combinations of FNR and CRP binding sites. The co‐dependent versus synergistic interaction of these activators and their requirement for known activating regions have been examined. The results demonstrate that FNR can co‐activate when located upstream at around −91 bp in combination with either FNR or CRP downstream. When FNR occupies the downstream site the promoter is co‐dependent on an upstream activator, but not when CRP occupies this site. Activating region 1 in CRP (defined by substitutions at residue H159) and its putative equivalent in FNR (defined by substitutions at S73) are mainly required in the upstream activator; the putative equivalent in FNR of activating region 3 of CRP (defined by substitutions at G85 and K52, respectively) is mainly required in the dimer which binds downstream. Activating region 1 of FNR is required only in the downstream subunit of the upstream activator in a promoter which is co‐dependent on two FNR dimers. These data suggest that both bound upstream and downstream activators interact with RNA polymerase to promote transcription, and that co‐dependence is determined by the nature of the activator plus the promoter context.
Regulation of anaerobic citrate metabolism in Klebsiella pneumoniae
Authors: Bott Michael , Meyer Margareta , Dimroth Peter
Published: March 2004

Three enzymes are specifically required for uptake and catabolism of citrate by Klebsiella pneumoniae under anaerobic conditions: a Na+‐dependent citrate carrier (CitS), citrate lyase (CitDEF), and the Na+ pump oxalo‐acetate decarboxylase (OadGAB). The corresponding genes are clustered on the chromosome, with the citCDEFG genes located upstream and divergent to the citS—oadGAB genes. We found that expression of citS from its native promoter in Escherichia coli requires the DNA region downstream of oadB. Nucleotide sequence analysis of this region revealed the presence of two adjacent genes, citA and citB, By sequence similarity, the predicted CitA and CitB proteins were identified as members of the two‐component regulatory systems. The sensor kinase CitA contained, in the N‐terminal half, two putative transmembrane helices which enclosed a presumably periplasmic domain of about 130 amino acids. The C‐terminal half of the response regulator CitB harboured a helix‐turn‐helix motif typical of DNA‐binding proteins. K. pneumoniaecitB null mutants were unable to grow anaerobically with citrate as the sole carbon and energy source (Cit− phenotype). When cultivated anaerobically with citrate plus glycerol, all of the citrate‐specific fermentation enzymes were synthesized in the wild type, but not in the citB mutants. This showed that citS, oadGAB and citDEF required the CitB protein for expression and therefore are part of a regulon. In the wild type, synthesis of CitS, oxalo‐acetate decarboxylase and citrate lyase was dependent on the presence of citrate, sodium ions and a low oxygen tension. In a citA null mutant which expressed citB constitutively at high levels, none of these signals was required for the formation of the citrate fermentation enzymes. This result suggested that citrate, Na+, and oxygen exerted their regulatory effects via the CitA/CitB system. In the presence of these signals, the citAB gene products induced their own synthesis. The positive autoregulation occurred via co‐transcription of citAB with citS and oadGAB.
Genetic and functional evidence that Type IV pili are required for social gliding motility in Myxococcus xanthus
Authors: Wu Samuel S. , Kaiser Dale
Published: March 2004

The social gliding behaviour of Myxococcus xanthus has previously been associated with the presence of polar pili. A Tn5 transposon insertion was isolated which introduces a defect in social gliding and is genetically linked to a known sgl locus; this insertion was found also to cause a piliation defect. A 2.7 kb section of DNA was isolated from either side of this transposon and sequenced, revealing three genes which encode amino acid sequences with substantial similarity to components of the Type IV pilus biogenesis pathway in Pseudomonas aeruginosa. The myxococcal pilA gene encodes a putative pilin precursor with a short signal sequence and processing site similar to those of other Type IV pilins. Myxococcal pilS and pilR encode amino acid sequences with similarity to PilS and PilR of P. aeruginosa, as well as to other members of the NtrB/C family of two‐component regulators. Mutations within pilR and pilA that have no polar effect were demonstrated to be responsible for pilus and social motility defects. These results indicate that the pili of M. xanthus belong to the Type IV family of pili, and demonstrate that these pili are actually required for social motility.