bluuebunny/crossref_metadata_2025 huggingface dataset:
A recombinational defect in the C‐terminal domain of Escherichia coli RecA2278‐5 protein is compensated by protein binding to ATP
Authors: Alexseyev Andrey A. , Baitin Dmitry M. , Kuramitsu Seiki , Ogawa Tomoko , Ogawa Hideyuki , Lanzov Vladislav A.
Published: November 2003

RecA2278‐5 is a mutant RecA protein (RecAmut) bearing two amino acid substitutions, Gly‐278 to Thr and Val‐275 to Phe, in the α‐helix H of the C‐terminal sub‐domain of the protein. recA2278‐5 mutant cells are unusual in that they are thermosensitive for recombination but almost normal for DNA repair of UV damage and the SOS response. Biochemical analysis of purified RecAmut protein revealed that its temperature sensitivity is suppressed by prior binding of this protein to its ligand. In fact, the preheating of RecAmut protein for several minutes at a restrictive temperature (42°C) in the absence of ATP resulted in inhibition at 42°C of many activities related to homologous recombination including ss‐ and dsDNA binding, high‐affinity binding for ATP, ss‐ or dsDNA‐dependent ATPase, RecA–RecA interaction, and strand transfer capability. The binary complex RecAmut::ATP under the same conditions showed a decrease in only two activities, i.e. dsDNA binding and high‐affinity binding for ATP. Besides ATP, sodium acetate (1.5M) was shown to be another factor that can stabilize the RecAmut protein at 42°C, judging by restoration of its DNA‐free ATPase activity. The similarity of influence of high salt (with its non‐specific binding) and ATP (binding specifically) on the apparent protein folding stability suggests that the structural stability of the RecA C‐terminal domain is one of the conditions for correct interaction between RecA protein and ATP in the RecA::ATP::ssDNA presynaptic complex formation. The decrease in affinity for ATP was suggested to be the factor that determined a particular recombinational (but not repair) thermosensitivity of the RecAmut protein. Finally, we show that the stability of C‐terminal domain appeared to be necessary for the dsDNA‐binding activity of the protein.
Replication control of plasmid pLS1: the antisense RNA II and the compact rnaII region are involved in translational regulation of the initiator RepB synthesis
Authors: Del Solar Gloria , Acebo Paloma , Espinosa Manuel
Published: November 2003

Replication of the streptococcal plasmid pLS1 is controlled by two plasmid‐encoded gene products: the repressor protein CopG and the antisense RNA, RNA II. Two different mutants in rnaII have been isolated. The 5′‐end and the levels of RNA II synthesized by pneumococcal cells harbouring the wild‐type pLS1 or mutant plasmids (affected in either genes copG or rnaII ) were analysed. One of the rnaII mutants exhibited a high‐copy‐number phenotype, whereas an in vitro‐constructed mutation, which affects the −10 region of the rnaII promoter, resulted in plasmids lacking copy‐number phenotype. The latter mutation had a pleiotropic effect: it abolished RNA II synthesis, but it also affected the initiation of translation signals of the gene encoding the RepB initiator protein. Transcriptional and translational fusions, together with in vitro inhibition of RepB synthesis by specific oligonucleotides, showed translational inhibition of RepB synthesis by RNA II, perhaps by directly blocking the accessibility of the ribosomes to the repB initiation of translation signals.
Sigma‐E is required for the production of the antibiotic actinomycin in Streptomyces antibioticus
Authors: Jones George H. , Paget Mark S. B. , Chamberlin Leony , Buttner Mark J.
Published: November 2003

The phsA gene encodes phenoxazinone synthase (PHS), which catalyses the penultimate step in the pathway for actinomycin biosynthesis in Streptomyces antibioticus. The phsA promoter strikingly resembles a putative StreptomycesσE cognate promoter, and purified EσE holoenzyme transcribed the phsA promoter in vitro. However, the phsA promoter was still active in an S. antibioticussigE null mutant and the level of PHS activity was unaffected. Despite this, disruption of sigE blocked actinomycin production completely. The loss of actinomycin production correlated with a 10‐fold decrease in the activity of actinomycin synthetase I, the enzyme which catalyses the activation of the precursor of the actinomycin chromophore.
Structure, function and immunogenicity of streptococcal antigen I/II polypeptides
Authors: Jenkinson Howard F. , Demuth Donald R.
Published: November 2003

The antigen I/II family of cell‐surface‐anchored polypeptides in oral streptococci are structurally complex multi‐functional adhesins, with multiple ligand‐binding sites. Discrete regions within these polypeptides bind human salivary glycoproteins, other microbial cells, and calcium. Sequences within the N‐terminal region bind preferentially fluid‐phase glycoproteins, while the C‐terminal half of the polypeptide contains species‐specific adhesion‐mediating sequences that bind surface‐immobilized glycoproteins. These features may assist streptococcal adhesion to oral surface receptors despite the presence of excess fluid‐phase receptors. Immunological studies reveal an array of T‐cell and B‐cell epitopes presented by antigen I/II polypeptides and suggest the occurrence of natural suppression of human antibodies to the adhesion‐mediating sequences. The functional and immunological properties of antigen I/II proteins may account to a major extent for the success of oral streptococci colonizing and surviving within the human host.
ADP‐ribosylation by the extracellular fibrils of Myxococcus xanthus
Authors: Hildebrandt Kim , Eastman Deborah , Dworkin Martin
Published: November 2003

The isolated, extracellular fibrils of the myxobacterium, Myxococcus xanthus, are capable of carrying out ADP‐ribosylation. The substrate for the ADP‐ribosylation is reactive with monoclonal antibody 2105, which has been shown to be directed specifically against the integral fibril proteins. The extracellular fibrils thus contain both the ADP‐ribosyl transferase and the substrate for the ribosylation. This process may play a role in the contact‐mediated cell–cell interactions that are an important part of the social behaviour of M. xanthus.
The roles of proteins L28 and L33 in the assembly and function of Escherichia coli ribosomes in vivo
Authors: Maguire Bruce A. , Wild Donald G.
Published: November 2003

Strain BM108 of Escherichia coli has a chromosomal mutation in the rpmB,G operon that prevents synthesis of ribosomal proteins L28 and L33. The mutation was lethal unless synthesis of protein L28 was induced from a plasmid. Without protein L28, RNA and protein synthesis were linear rather than exponential. No 70S ribosomes were made. Instead, RNA accumulated in ‘30S material’ and ‘47S particles’; the latter were distinct from 50S ribosomal subunits, lacked proteins L28 and L33 and had substoicheometric amounts of three other proteins. When L28 synthesis was induced (but protein L33 was still absent), the strain grew as well as, and assembled 70S ribosomes with similar kinetics to, a wild‐type control. Thus, protein L28 is required for ribosome assembly in strain BM108 while protein L33 has no significant effect on ribosome synthesis or function.
DivIB, FtsZ and cell division in Bacillus subtilis
Authors: Rowland S. L. , Katis V. L. , Partridge S. R. , Wake R. G.
Published: November 2003

The Bacillus subtilis cell‐division protein DivIB is shown to be present at an ≈100‐fold higher abundance (≈5000 molecules per cell) than its Escherichia coli FtsQ homologue. B. subtilis contains much more DivIB (at least 60‐fold) than is needed to maintain the normal rate of cell division at moderate temperatures (up to 37°C). However, a high level of DivIB is needed to achieve the normal rate of division at high temperature (47°C). It is proposed that membrane‐bound DivIB is involved in stabilizing or promoting the assembly of the division complex (which is intrinsically temperature sensitive) in a manner that requires more of the protein at higher temperatures. The (at least) 60‐fold accumulation of DivIB and FtsZ from an undetectable level, following germination and outgrowth of spores up until the stage of the first cell division, was unaffected by blocking of initiation of the first round of replication. It is concluded that there is no major synthesis of either of these ‘division initiation’ proteins linked to initiation, progression or completion of the first round of replication accompanying spore outgrowth.
Mutational analysis of receptor binding mediated by the Dr family of Escherichia coli adhesins
Authors: Carnoy Christophe , Moseley Steve L.
Published: November 2003

The fimbrial and afimbrial adhesins of the Dr family mediate the adherence of uropathogenic and diarrhoea‐associated Escherichia coli to decay‐accelerating factor (DAF) present on erythrocytes and other cell types. The Dr haemagglutinin binds type IV collagen and, unlike other members of the Dr family, mediates an adherence inhibited in the presence of chloramphenicol. We examined the ability of other members of the Dr family—AFAI, AFAIII, and F1845—to bind to type IV collagen, and demonstrated that the collagen‐binding phenotype was unique to the Dr haemagglutinin. We employed site‐directed mutagenesis to demonstrate the requirement of a negatively charged amino‐acid at position 54 of the Dr haemagglutinin subunit for chloramphenicol sensitivity of binding. Mutations at position 32, 40, 54, 90, and 113 differently affected type IV collagen binding and chloramphenicol sensitivity of binding, while retaining DAF‐binding capability. These results suggest the existence of a conformational receptor‐binding domain in the major structural subunit of Dr family adhesins and demonstrate that chloramphenicol sensitivity of binding and adherence to type IV collagen were independent and separable phenotypes. Finally, we showed that the two conserved cysteine residues of Dr family structural subunits form a disulphide bond and that mutations of these residues abolish haemagglutination and binding to type IV collagen.
Mycobacteriumbovis BCG genes involved in the biosynthesis of cyclopropyl keto‐ and hydroxy‐mycolic acids
Authors: Dubnau Eugenie , Lanéelle Marie‐Antoinette , Soares Sonia , Bénichou Anne , Vaz Tania , Promé Danielle , Promé Jean‐Claude , Daffé Mamadou , Quémard Annai¨k
Published: November 2003

The resurgence of tuberculosis and the emergence of multidrug‐resistant mycobacteria necessitate the development of new antituberculosis drugs. The biosynthesis of mycolic acids, essential elements of the mycobacterial envelope, is a good target for chemotherapy. Species of the Mycobacterium tuberculosis complex synthesize oxygenated mycolic acids with keto and methoxy functions. In contrast, the fast‐growing Mycobacterium smegmatis synthesizes oxygenated mycolic acids with an epoxy function. We describe the isolation and sequencing of a cluster of four genes from Mycobacterium bovis bacillus Calmette–Guérin (BCG), coding for methyl transferases, and which, when transferred into M. smegmatis,allow the synthesis of ketomycolic acid, in addition to an as yet undescribed mycolic acid, hydroxymycolic acid. These oxygenated mycolic acids, unlike the regular mycolic acids of M. smegmatis, and similar to the mycolic acids of M. bovis, are highly cyclopropanated. Furthermore, there is a perfect match between the structures of the keto‐ and the hydroxy‐mycolic acids. We propose a biosynthetic model in which there is a direct relationship between these two types of mycolic acid.
A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K‐12
Authors: McDaniel Timothy K. , Kaper James B.
Published: November 2003

Attaching and effacing (AE) bacteria are a diverse group of gastrointestinal pathogens, comprising members of four genera, that cause the intestinal epithelial microvilli to be replaced with raised clusters of filamentous actin that conform to the surface of attached bacteria. We have cloned a 35.4 kb ‘pathogenicity island’ from the prototype AE bacterium, enteropathogenic Escherichia coli, containing all previously described AE genes. Transfer of this pathogenicity island to avirulent E. coli converts the recipients into strains that secrete virulence proteins, induce host signal‐transduction pathways, and cause AE lesions on cultured epithelial cells. These results demonstrate that this pathogenicity island contains all pathogen‐specific genes necessary for inducing AE lesions, and that the defining feature of this class of pathogens can be acquired by an avirulent bacterium in a single genetic step.
Transcription induction of the ferric citrate transport genes via the N‐terminus of the FecA outer membrane protein, the Ton system and the electrochemical potential of the cytoplasmic membrane
Authors: Kim InSook , Stiefel Alfred , Planto¨r Stefan , Angerer Annemarie , Braun Volkmar
Published: November 2003

Ferric citrate induces transcription of the ferric citrate transport genes fecABCDE without entering the cells of Escherichia coli K‐12. Point mutants of the outer membrane‐receptor protein FecA are affected in induction independent of the FecA transport activity, suggesting that FecA is directly involved in induction. Alignment of FecA with the other ferric siderophore receptors of E. coli reveals an N‐terminal extension in FecA that is not found in the receptors whose synthesis is not induced by their cognate ferric siderophores. In this study, we show that excision of the N‐terminal region abolished the inducing activity of FecA, but retained its transport activity. Overproduction of the N‐terminal FecA fragment inhibited FecA‐dependent induction, but not transport. Constitutive expression caused by C‐terminally truncated FecR derivatives was not inhibited by the N‐terminal FecA fragment. The N‐terminal region of FecA was localized in the periplasm, which indicates that FecA probably interacts with FecR, which is involved in signal transduction across the cytoplasmic membrane. Transcription initiation of the fec transport genes required the Ton system, consisting of TonB, ExbB, and ExbD, and was inhibited by carbonylcyanide‐m‐chlorophenylhydrazone (CCCP) and 2,4‐dinitrophenol (DNP), which dissipate the electrochemical potential of the cytoplasmic membrane. fec transcription of mutant fecA4, which displays constitutive fec transcription in the absence of TonB, was not affected by CCCP. The data support a model that proposes initiation of fec transport gene transcription by binding of ferric citrate to FecA. The transcription initiation signal is transferred across the outer membrane through the activity of the Ton system at the expense of the electrochemical potential of the cytoplasmic membrane. The N‐terminus of FecA interacts in the periplasm with the C‐terminus of FecR, through which the signal is transferred across the cytoplasmic membrane into the cytoplasm, where it increases the activity of the sigma factor FecI, which then directs the RNA polymerase to the fec promoter upstream of fecA.
The com locus controls genetic transformation in Streptococcus pneumoniae
Authors: Cheng Q. , Campbell E. A. , Naughton A. M. , Johnson S. , Masure H. R.
Published: November 2003

Genetic exchange by natural transformation in Streptococcus pneumoniae occurs in a cell‐density dependent process and is initiated by a small extracellular signalling molecule, the competence‐stimulating peptide (CSP). comC, the gene for this peptide, has previously been identified and encodes a 44 amino acid pre‐peptide that is apparently processed to an active molecule that consists of the C‐terminal 17 amino acids. We have sequenced the region adjacent to comC and shown that it is the first gene of an operon, com, consisting of two downstream elements, comD and comE, which encode members of the two‐component family of sensor regulators. Null mutants with defects in either comC or comD were transformation deficient and failed to respond to exogenous CSP. A comC mutant did not exhibit any detectable CSP activity, while a comD mutant that contained an intact comC produced minimal CSP activity. In mixed‐culture experiments consisting of isogenic pairs of pneumococci (Csp+ and Csp−), we showed that induction of competence by quorum sensing was independent of CSP. Northern analysis showed that com was transcribed as a single polycistronic message, while analysis of strains with transcriptional fusions showed that com was constitutively expressed under conditions that both promoted or repressed the development of competence. Finally, we showed genetically and biochemically a CSP‐dependent transcription of rec, a competence‐induced locus, and that ComD and ComE are required for this CSP‐dependent expression.
PilP, a pilus biogenesis lipoprotein in Neisseria gonorrhoeae, affects expression of PilQ as a high‐molecular‐mass multimer
Authors: Drake Sandra L. , Sandstedt Sara A. , Koomey Michael
Published: November 2003

Studies of gonococcal pilus biogenesis are fundamental to understanding organelle structure/function relationships and identifying new approaches to controlling disease. This area of research is also relevant to elucidating the basic mechanisms of outer membrane translocation of macromolecules, which requires components highly related to those involved in type IV pilus expression. Previous studies have shown that products of several ancillary pil genes are required for organelle biogenesis but of these only PilQ, a member of the GspD protein family, is a component of the outer membrane. DNA sequencing of the region upstream of pilQ revealed the presence of two open reading frames (ORFs) whose deduced polypeptides shared significant identities with proteins required for pilus expression in Pseudomonas aeruginosa and Pseudomonas syringae, the genes for which are arrayed upstream of a gene encoding a PilQ homologue. Gonococcal mutants bearing transposon insertions in these ORFs were non‐piliated and failed to express pilus‐associated phenotypes, and the corresponding genes were designated pilO and pilP. The piliation defects in the mutants could not be ascribed to polarity on distal pilQ expression as shown by direct measurement of PilQ antigen in those backgrounds and the use of a novel technique to create tandem duplications in the gonococcus (Gc) genome. As predicted by the presence of a consensus lipoprotein signal sequence, PilP expressed in both Escherichia coli and Gc could be labelled with [3H]‐palmitic acid. PilP− as well as PilQ− mutants shed PilC, a protein which facilitates pilus assembly and is implicated in epithelial cell adherence, in a soluble form. Combined with the finding that levels of multimerized PilQ were greatly reduced in PilP− mutants, the results suggest that PilP is required for PilQ function and that PilQ and PilC may interact during the terminal stages of pilus biogenesis. The findings also support the hypothesis that the Gc PilQ multimer corresponds to a physiologically relevant form of the protein required for pilus biogenesis.
The dual role of Apl in prophage induction of coliphage 186
Authors: Reed Michael R. , Shearwin Keith E. , Pell Linda M. , Egan J. Barry
Published: November 2003

In the present study we show that the Apl protein of the temperate coliphage 186 combines, in one protein, the activities of the coliphage lambda proteins Cro and Xis. We have shown previously that Apl represses both the lysogenic promoter, pL, and the major lytic promoter, pR, and is required for excision of the prophage. Apl binds at two locations on the phage chromosome, i.e. between pR and pL and at the phage‐attachment site. Using an in vivo recombination assay, we now show that the role of ApI in excision is in the process itself and is not simply a consequence of repression of pR or pL. To study the repressive role of Apl at the switch promoters we isolated Apl‐resistant operator mutants and used them to demonstrate a requirement for Apl in the efficient derepression of the lysogenic promoter during prophage induction. We conclude that Apl is both an excisionase and transcriptional repressor.
Characterization of the locus encoding the Streptococcus pneumoniae type 19F capsular polysaccharide biosynthetic pathway
Authors: Morona Judy K. , Morona Renato , Paton James C.
Published: November 2003

We have previously reported the nucleotide sequence of the first six genes of the Streptococcuspneumoniae type 19F capsular polysaccharide biosynthesis locus (cps19f). In this study we used plasmid insertion/rescue and inverse polymerase chain reaction (PCR) to clone an additional 10 kb downstream region containing the remainder of the cps19f locus, which was then subjected to sequence analysis. The cps19f locus is located in the S. pneumoniae chromosome between dexB and aliA, and consists of 15 open reading frames (ORFs), designated cps19fA to cps19fO, that appear to be arranged as a single transcriptional unit. Insertion‐duplication mutants in seven out of the nine new ORFs have been constructed in a smooth type 19F strain, all of which resulted in a rough (non‐encapsulated) phenotype, confirming that the operon is essential for capsule production. Comparison with sequence databases has allowed us to propose functions for 12 of the cps19f gene products, and a biosynthetic pathway for type 19F capsular polysaccharide. T7 expression studies confirmed that cps19fH, cps19fK, cps19fL, cps19fM and cps19fN directed the production of polypeptides of the expected size in Escherichia coli. The function of the cps19fK product was confirmed by its ability to complement a mutation in nfrC (rffE ) in E. coli, as judged by restoration of sensitivity to bacteriophage N4. Interestingly, the last four genes of the locus (cps19fL–O ) exhibit very strong homology (up to 70% amino acid identity) to a portion of the Shigella flexneri rfb gene cluster encoding biosynthesis of dTDP‐rhamnose. When expressed in E. coli, cps19fL–O were capable of complementing a mutation deleting the respective Shigella flexneri homologues. Southern hybridization analysis indicated that cps19fA and cps19fB were the only cps genes found in all 16 S. pneumoniae serotypes/groups tested. The region from cps19fG to cps19fK was found only in members of serogroup 19, and, within this, cps19fI was unique to type 19F.
Modulation of the surface architecture of Gram‐negative bacteria by the action of surface polymer:lipid A–core ligase and by determinants of polymer chain length
Authors: Whitfield Chris , Amor Paul A. , Ko¨plin Reinhard
Published: November 2003

Lipopolysaccharides (LPSs) are complex glycolipids found in the outer membrane of Gram‐negative bacteria. The lipid A–core component of the LPS molecule provides a versatile anchor to which a surface polymer:lipid A–core ligase enzyme can attach one or more structurally distinct surface polymers in a single bacterial strain. In some cases the same polymer can be found on the cell surface in both lipid A–core‐linked and ‐unlinked forms. Analysis by SDS–PAGE of populations of LPS molecules extracted from bacterial cells indicates that there is extensive heterogeneity in their size distribution. Much of the heterogeneity results from complex modal distributions in the chain length of the polymers which are attached to lipid A–core. This is the result of preferential ligation of polymers with specific degrees of polymerization during the assembly of the LPS molecule. The surface architecture of the Gram‐negative bacterial cell is therefore profoundly affected by the activities of the surface polymer:lipid A–core ligase and by molecular determinants of polymer chain length. Because of the involvement of cell‐surface polymers in interactions between pathogenic bacteria and their hosts, these enzymatic activities also have an important impact on virulence. In this review, the organization of LPSs and related surface polymers will be described and the current understanding of the molecular mechanisms involved in surface diversity will be discussed. Emphasis is placed on the Enterobacteriaceae, but similarities to other bacteria suggest that aspects of the enterobacterial system will have broader significance.
Haem iron‐transport system in enterohaemorrhagic Escherichia coli O157:H7
Authors: Torres Alfredo G. , Payne Shelley M.
Published: November 2003

In this study, we identified the iron‐transport systems of Escherichia coli O157:H7 strain EDL933. This strain synthesized and transported enterobactin and had a ferric citrate transport system but lacked the ability to produce or use aerobactin. It used haem and haemoglobin, but not transferrin or lactoferrin, as iron sources. We cloned the gene encoding an iron‐regulated haem‐transport protein and showed that this E. coli haem‐utilization gene (chuA) encoded a 69 kDa outer membrane protein that was synthesized in response to iron limitation. Expression of this protein in a laboratory strain of E. coli was sufficient for utilization of haem or haemoglobin as iron sources. Mutation of the chromosomal chuA and tonB genes in E. coli O157:H7 demonstrated that the utilization of haemin and haemoglobin was ChuA‐ and TonB‐dependent. Nucleotide sequence analysis of chuA revealed features characteristic of TonB‐dependentFur‐regulated, outer membrane iron‐transport proteins. It was highly homologous to the shuA gene of Shigella dysenteriae and less closely related to hemR of Yersinia enterocolitica and hmuR of Yersinia pestis. A conserved Fur box was identified upstream of the chuA gene, and regulation by Fur was confirmed.
Carbon‐source regulation of virulence gene expression in Listeria monocytogenes
Authors: Milenbachs Andrea A. , Brown David P. , Moors Marlena , Youngman Philip
Published: November 2003

All known virulence genes of Listeria monocytogenes are under positive regulation by the transcription factor PrfA. Previous work employing the L. monocytogenes strain NCTC7973 suggested that the disaccharide cellobiose might serve as a specific ‘signature molecule’ which functions to prevent activation of the PrfA‐controlled regulon in a soil environment. We have examined three other L. monocytogenes strains, 10403S, LO28 and EGD, all commonly regarded as wild‐type isolates, and find that NCTC7973 is anomalous with respect to the effect of carbohydrates on the expression of PrfA‐controlled gene expression. In the case of 10403S, LO28 and EGD, several other readily metabolized mono‐ and disaccharides are as effective as cellobiose in repressing expression of the PrfA‐controlled gene hly, indicating that the cellobiose effect is not specific, and suggesting that NCTC7973 may be a partially deregulated variant. Moreover, concentrations of cellobiose and other sugars required for repression of hly expression (> 1 mM) were found to significantly enhance growth of L. monocytogenes cultures, suggesting that the repression phenomenon probably results from a metabolic effect of sugar utilization rather than a signal‐sensing response. Thus the previously reported cellobiose effect may reflect an aspect of a more global mechanism of catabolite repression in L. monocytogenes. Although cellobiose represses expression of hly and plcA at the level of transcript accumulation, quantitative Western blot analysis indicates that cellobiose has no effect on PrfA levels. These results are consistent with a model in which PrfA activity is controlled by interaction with a hypothetical cofactor, the synthesis or depletion of which is responsive to the presence of readily metabolized carbohydrates.
HoxA is a transcriptional regulator for expression of the hup structural genes in free‐living Bradyrhizobium japonicum
Authors: Van Soom C. , De Wilde P. , Vanderleyden J.
Published: November 2003

A chromosomally integrated Bradyrhizobium japonicum hoxA mutant is unable to oxidize hydrogen in free‐living conditions. Derepressing conditions that induce hydrogenase activity in free‐living, wild‐type B. japonicum cells cannot induce expression of the hydrogenase structural genes in the hoxA mutant. The DNA‐binding capacity of HoxA at the hup promoter region was studied by means of gel retardation. Both heterotrophically growing cells and cells induced to express hydrogenase activity contain a protein that specifically binds to the hup promoter region. Crude protein extracts isolated from a B. japonicum hoxA mutant do not contain this binding compound. The HoxA protein was overexpressed in E. coli and isolated in the form of a maltose‐binding protein (MBP)–HoxA fusion. The MBP–HoxA hybrid protein specifically bound to a 50 bp region of the hupSL promoter known to be important for regulation of hupSL expression.
In vivo phase variation of MR/P fimbrial gene expression in Proteus mirabilis infecting the urinary tract
Authors: Zhao Hui , Li Xin , Johnson David E. , Blomfield Ian , Mobley Harry L. T.
Published: November 2003

Proteus mirabilis, associated with complicated urinary tract infection, expresses mannose‐resistant/Proteus‐like (MR/P) fimbriae. Expression of these surface structures, which mediate haemagglutination and have a demonstrated role in virulence, undergoes phase variation. By DNA sequence analysis, a 252 bp invertible element was found in the intergenic region between mrpI, the putative site‐specific recombinase gene, and mrpA, the primary structural subunit gene. The invertible segment is flanked by identical 21 bp inverted repeats and the presumptive half‐sites for recombinase binding show homology to those recognized by FimB and FimE encoded by the Escherichia colifim (Type 1 fimbriae) gene cluster. When amplified by the polymerase chain reaction (PCR) from static broth cultures expressing MR/P fimbriae, the switch region was found in both ON and OFF positions. When PCR was used to amplify agar cultures which do not express the fimbriae, the switch region was OFF only. A canonical σ70 promoter inside the invertible element drives the transcription of mrpA when in the ON position; in the OFF position it is directed away from mrpA but does not appear to drive expression of mrpI. The mrpI gene was able to confer inversion of the mrp switch region in trans from both ON to OFF and OFF to ON. To examine the position of the switch in vivo, urine, bladder, and kidneys from mice transurethrally infected with P. mirabilis were used to prepare template DNA for PCR amplification. In the absence of urolithiasis (urease‐mediated stone formation), the switch was found 100% in the ON position, a condition never observed following in vitro culture. We conclude that MR/P phase variation is regulated at the transcriptional level by the action of MrpI on an invertible element and that there is strong selective pressure for the expression of MR/P fimbriae in vivo.
The prpR1 and prR2 arginine‐specific protease genes of Porphyromonas gingivalis W50 produce five biochemically distinct enzymes
Authors: Rangarajan Minnie , Aduse‐Opoku Joseph , Slaney Jennifer M. , Young Katherine A. , Curtis Michael A.
Published: November 2003

The arginine‐specific protease activity of Porphyromonas gingivalis is considered to be an important factor in the pathogenic potential of this organism in destructive periodontal disease. Multiple forms of closely related Arg‐x proteases are present in the culture supernatants of P. gingivalis W50. RI is a heterodimer (α/β) in which the catalytic α chain is associated with a second β chain which functions as a haemagglutinin. RIA is a single‐chain enzyme (α) and RIB is a highly post‐translationally lipid‐modified enzyme (LPS‐α) with reduced solubility compared to the other two forms. The N‐terminal sequence of the α chain of all three forms is identical, suggesting that all these enzymes may arise by differential processing of the prpR1 (protease polyprotein for RI). In the present study we constructed a prpR1− strain of P. gingivalis W50 by insertional gene inactivation and characterized the residual extracellular Arg‐x protease activity of the resulting mutant. Loss of prpR1 expression led to the abolition of RI, RIA and RIB but the total Arg‐x activity in the supernatant of this strain was reduced by only c. 66%. The remaining activity was composed of two novel forms of Arg‐x protease (RIIA and RIIB) which appeared to be structurally and kinetically almost identical to RIA and RIB, respectively, except for two amino acid differences in the N‐terminus at position 8 (Q→E) and position 17 (A→P) and with respect to their stability to high pH. Confirmation that RIIA and RIIB are the products of a homologous locus (prR2) was obtained by cloning and sequencing the prR2 which showed the predicted substitutions in the deduced translation. These data indicate that RI, RIA and RIB are produced by prpR1 expression and a maturation pathway which can give rise to a dimer and an unmodifed‐ or LPS‐modified catalytic monomer. Furthermore, RIIA and RIIB, the products of prR2, are exported into the culture supernatant in the absence of prpR1 expression and these forms may also contribute to the pathogenic potential of this organism in destructive disease.
Regulation of expression, genetic organization and substrate specificity of xylose uptake in Bacillus megaterium
Authors: Schmiedel Dagmar , Kintrup Martin , Ku¨ster Elke , Hillen Wolfgang
Published: November 2003

Xylose uptake in Bacillus megaterium depends on expression of a putative H+/xylose symporter encoded by xylT, the last gene in the xyl operon. Insertional inactivation of xylT leads to an apparent uptake deficiency determined with whole cells and severely slower growth on xylose as sole carbon source. Expression of XylT is xylose inducible and subject to carbon catabolite repression mediated by CcpA and cre. Northern analysis of the xyl mRNA reveals that a potential stem‐loop structure located in the non‐translated region between xylA and xylB presumably acts as a transcriptional terminator, as it leads to different amounts of the respective mRNA sections: the 5′‐xylA portion is very abundant, while the 3′‐xylBT portion constitutes only a fraction of it. XylT has an apparent Michaelis constant (KM) of approx. 100 μM and is competitively inhibited by glucose with an inhibitor constant KI of 16 mM.
Catabolite repression of the Bacillus subtilis gnt operon exerted by two catabolite‐responsive elements
Authors: Miwa. Yasuhiko , Nagura Kazuya , Eguchi Susumu , Fukuda Hirokazu , Deutscher Josef , Fujita Yasutaro
Published: November 2003

Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis‐acting catabolite‐responsive element (cre) by a complex of CcpA, which is a member of the GalR‐LacI family of bacterial regulatory proteins, and the seryl‐phos‐phorylated form of HPr (P‐ser‐HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the ere in the first gene (gntR) of the gnt operon (credown), this operon contains another ere located in the promoter region (creup). A translational gntR‐lacZ fusion expressed under the control of various combinations of wild‐type and mutant credown and creup was integrated into the chromosomal amyE locus, and then catabolite repression of p‐galac‐tosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by creup was probably independent of catabolite repression exerted by credown; both creup and credown catabolite repression involved CcpA. Catabolite repression exerted by creup was independent of P‐ser‐HPr, and catabolite repression exerted by credown was partially independent of P‐ser‐HPr. DNase I footprinting experiments indicated that a complex of CcpA and P‐ser‐HPr did not recognize creup, in contrast to its specific recognition of credown. However, CcpA complexed with glucose‐6‐phosphate specifically recognized creup as well as credown, but the physiological significance of this complexing is unknown.
Molecular characterization of the Bacillus anthracis main S‐layer component: evidence that it is the major cell‐associated antigen
Authors: Mesnage Stéphane , Tosi‐Couture Evelyne , Mock Michèle , Gounon Pierre , Fouet Agnès
Published: November 2003

Bacillus anthracis, the aetiological agent of anthrax, is a Gram‐positive spore‐forming bacterium. The cell wall of vegetative cells of B. anthracis is surrounded by an S‐layer. An array remained when sap, a gene described as encoding an S‐layer component, was deleted. The remaining S‐layer component, termed EA1, is chromosomally encoded. The gene encoding EA1 (eag) was obtained on two overlapping fragments in Escherichia coli and shown to be contiguous to the sap gene. The EA1 amino acid sequence, deduced from the eag nucleotide sequence, shows classical S‐layer protein features (no cysteine, only 0.1% methionine, 10% lysine, and a weakly acidic pi). Similar to Sap and other Gram‐positive surface proteins, EA1 has three 'S‐layer‐homology’motifs immediately downstream from a signal peptide. Single‐ and double‐disrupted mutants were constructed. EA1 and Sap were co‐localized at the cell surface of the wild‐type bacilli. However, EA1 was more tightly bound than Sap to the bacteria. Electron microscopy studies and in vivo experiments with the constructed mutants showed that EA1 constitutes the main lattice of the B. anthracis S‐layer, and is the major cell‐associated antigen.
A two‐gene ABC‐type transport system that extrudes Na+ in Bacillus subtilis is induced by ethanol or protonophore
Authors: Cheng Jianbo , Guffanti Arthur A. , Krulwich Terry Ann
Published: November 2003

A transposition mutant of Bacillus subtilis (designated JC901) that was isolated on the basis of growth inhibition by Na at elevated pH, was deficient in energy‐dependent Na extrusion. The capacity of the mutant JC901 for Na ‐dependent pH homeostasis was unaffected relative to the wild‐type strain, as assessed by regulation of cytoplasmic pH after an alkaline shift. The site of transposition was near the 3 ‐terminal end of a gene, natB, predicted to encode a membrane protein, NatB. NatB possesses six putative membrane‐spanning regions at its C‐terminus, and exhibits modest sequence similarity to regions of eukaryotic Na+/H+ exchangers. Sequence and Northern blot analyses suggested that natB forms an operon with an upstream gene, natA. The predicted product of natA is a member of the family of ATP‐binding proteins that are components of transport systems of the ATP‐binding cassette (ABC) or traffic ATPase type. Expression of the lacZ gene that was under control of the promoter for natAB indicated that expression of the operon was induced by ethanol and the protonophore carbonylcyanide p‐chlorophenylhydrazone (CCCP), and, more modestly, by Na+, and K+, but not by choline or a high concentration of sucrose. Restoration of the natAB genes, cloned in a recombinant plasmid (pJY1), complemented the Na+‐sensitive phe‐notype of the mutant JC901 at elevated pH and significantly increased the resistance of the mutant to growth inhibition by ethanol and CCCP at pH 7; ethanol was not excluded, however, from the cells expressing natAB, so ethanol‐resistance does not result from NatAB‐dependent ethanol efflux. Transformation of the mutant with pJY1 did markedly enhance the capacity for Na+
DnaA protein stimulates polA gene expression in Escherichia coli
Authors: Quiñones Ariel , Wandt Gudrun , Kleinstäuber Sabine , Messer Walter
Published: November 2003

The polA gene of Escherichia coli encodes DNA polymerase I that is involved in DNA replication and repair. Despite the wide knowledge about structure and function of DNA polymerase I, there is little insight into the regulatory mechanisms involved in polA expression. DnaA is the initiator protein for DNA replication in E. coli. There are two putative DnaA‐binding sites within the extended promoter region of polA. In this work we studied the influence of altered levels of DnaA protein on polA expression. We found that DnaA overproduction increases polA expression in stationary‐phase cultures. The stimulation effect was independent of rpoS, which encodes the sigma factor for stationary‐phase‐inducible genes. However, it was modulated by ppGpp. Comparative S1 analyses revealed that the induction was based on transcriptional stimulation. Footprint‐ing experiments demonstrated that DnaA binds only to the proximal DnaA box near the polA promoter. These results suggest an additional role for DnaA as transcriptional activator of polA at least under certain physiological conditions.
Random shuttle mutagenesis: gonococcal mutants deficient in pilin antigenic variation
Authors: WIehr Ian J. , Seifert H. Steven
Published: November 2003

Shuttle mutagenesis has been adapted to randomly mutate the genome of Neisseria gonorrhoeae (gono‐coccus; Gc). A size‐restricted plasmid library of Gc strain FA1090 was mutated with the mini‐transposon mTnEGNS. Randomness was tested by checking for transposon insertion bias between vector and insert DNA, Gc transformation efficiency of individual mutated clones, and representation of unique clones before and after Gc transformation with a mutated pool of DNA. Mutants created by random shuttle mutagenesis were screened, using a colony‐based polymerase chain reaction assay, for the ability to undergo pilin antigenic variation. Out of 8064 mutants screened, 22 unique transposon insertion mutants were found to be antigenic variation deficient (Avd). The Avd mutants were separated into five types according to recombination defect‐associated phenotypes, including colony growth, natural DNA transformation competence, and repair of DNA damage caused by ultraviolet radiation.
Maltose‐binding protein interacts simultaneously and asymmetrically with both subunits of the Tar chemoreceptor
Authors: Gardina Paul J. , Bormans Arjan F. , Hawkins Murphy A. , Meeker Joshua W. , Manson Michael D.
Published: November 2003

The Tar chemotactic signal transducer of Escherichia coli mediates attractant responses to L‐aspartate and to maltose. Aspartate binds across the subunit interface of the periplasmic receptor domain of a Tar homodimer. Maltose, in contrast, first binds to the periplasmic maltose‐binding protein (MBP), which in its ligand‐stabilized closed form then interacts with Tar. Intragenic complementation was used to determine the MBP‐binding site on the Tar dimer. Mutations causing certain substitutions at residues Tyr‐143, Asn‐145, Gly‐147, Tyr‐149, and Phe‐150 of Tar lead to severe defects in maltose chemotaxis, as do certain mutations affecting residues Arg‐73, Met‐76, Asp‐77, and Ser‐83. These two sets of mutations defined two complementation groups when the defective proteins were co‐expressed at equal levels from compatible plasmids. We conclude that MBP contacts both subunits of the Tar dimer simultaneously and asymmetrically. Mutations affecting Met‐75 could not be complemented, suggesting that this residue is important for association of MBP with each subunit of the Tar dimer. When the residues involved in interaction with MBP were mapped onto the crystal structure of the Tar periplasmic domain, they localized to a groove at the membrane‐distal apex of the domain and also extended onto one shoulder of the apical region.
Gzf3p, a fourth GATA factor involved in nitrogen‐regulated transcription in Saccharomyces cerevisiae
Authors: Soussi‐Boudekou Saïd , Vissers Stephan , Urrestarazu Antonio , Jauniaux Jean‐Claude , André Bruno
Published: November 2003

In Saccharomyces cerevisiae, two positive transcription factors of the GATA family, Gln3p and NiMp/ Gatlp, upregulate the expression of multiple nitrogen pathway genes via upstream 5‐GATA‐3′ sequences. Another GATA factor, Uga43p/Da180p, downregulates to varying degrees the expression of some nitrogen‐regulated genes. Here, we report the functional analysis of a fourth GATA factor, Gzf 3p/Ni12p, whose gene was discovered by systematic sequencing of chromosome X. The Gzf3 protein most closely resembles Uga43p. Similar to Uga43p, Gzf3p has the properties of a negative GATA factor. While Uga43p is active specifically under nitrogen‐derepression conditions, Gzf 3p exerts its negative regulatory function specifically on preferred nitrogen sources: it is involved in nitrogen repression of NiMp‐dependent transcription. At least one positive GATA factor is required for the UGA43 and GZF3 genes to be expressed. The Uga43p factor negatively regulates GZF3 expression and vice versa. In addition, both Uga43p and Gzf3p moderately regulate expression of their own genes. These two proteins seem to be parts of a complex network of GATA factors which probably play a determining role in nitrogen‐regulated transcription.
Temperature‐regulated mRNA accumulation and stabilization for fatty acid desaturase genes in the cyanobacterium Synechococcus sp. strain PCC 7002
Authors: Sakamoto Toshio , Bryant Donald A.
Published: November 2003

Cyanobacteria acclimate to low‐temperature conditions by desaturating their membrane lipids. The desB (ω3 desaturase) and desC (A9 desaturase) genes of Synechococcus sp. strain PCC 7002 were cloned and characterized, and the expression of the desA (Δ12 desaturase), desB and desC genes was studied as a function of temperature. The steady‐state mRNA abundance for the desA gene was threefold higher in cells grown at 22 C than in cells grown at 38°C. des B transcripts were not detected at 38°C, but were abundant in cells grown at 22°C. Levels of desC mRNA were similar at both growth temperatures. The mRNA levels of each desaturase gene increased within 15min of a temperature shift‐down to 22°C, and mRNA levels recovered within 15min after a shift‐up to 38°C. The cold‐induced accumulation of transcripts from the desA and desB genes was suppressed by the addition of chloramphenicol, but the transient elevation of the desC transcript levels at 22°C was not affected by chloramphenicol. The half‐lives of the desA and desB mRNAs were significantly longer in cells grown at 22°C than in cells grown at 38°C, but the desC mRNA had a similar half‐life at both temperatures. These studies reveal three patterns of temperature regulation for the desaturase genes, whose expression is tightly controlled by a combination of mRNA synthesis and stabilization. These studies demonstrate that elevation of desaturase mRNA levels is not the rate‐limiting event during the low‐temperature acclimation of cyanobacteria.
Cell‐type specificity of the Anabaena fdxN‐element rearrangement requires xisH and xisl
Authors: Ramaswamy K. S. , Carrasco Claudio D. , Fatma Tasneem , Golden James W.
Published: November 2003

The fdxN element, along with two other DNA elements, is excised from the chromosome during heterocyst differentiation in Anabaena sp. strain PCC 7120. Previous work showed that rearrangement of the fdxN element requires the xisF gene, which encodes a site‐specific recombinase, and suggested that at least one other heterocyst‐specific factor is involved. Here we report that the xisH and xisl genes are necessary for the heterocyst‐specific excision of the fdxN element. Deletion of a 3.2 kb region downstream of the xisF gene blocked the fdxN‐element rearrangement in hetero‐cysts. The 3.2 kb deletion was complemented by the two overlapping genes xisH and xisl. Interestingly, extra copies of xlsHI on a replicating plasmid resulted in the xisF‐dependent excision of the fdxN element in vegetative cells. Therefore, xisHI are involved in the control of cell‐type specificity of the fdxN rearrangement. The xisHI genes had no effect on the two other DNA rearrangements. The xisHl‐induced excision of the fdxN element produced strains lacking the element and demonstrates that the 55 kb element contains no essential genes. xisH and xisl do not show similarity to any known genes.