bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characterization of catalase from Bordetella pertussis: identification of the katA promoter in an upstream insertion sequence
Authors: DeShazer David , Wood Gwendolyn E. , Friedman Richard L.
Published: October 2006

In this report we evaluate the role of catalase in the survival of Bordetella pertussis within human polymorphonuclear leukocytes (PMNs). Crude extracts of B. pertussis exhibited a single catalase activity when subjected to non‐denaturing polyacrylamide gel electrophoresis and assayed for catalase activity. A plasmid containing B. pertussis katA was identified by complementation of UM255, a catalase‐deficient strain of Escherichia coli. The nucleotide sequence of katA predicts a 55kDa protein that shares homology with a class of haem‐containing catalases found in both eubacteria and eukaryotes. Analysis of the nucleotide sequence upstream of katA revealed the presence of a copy of IS481, a B. pertussis‐specific insertion sequence. The start site of transcription of katA was mapped to a T residue in IS481 by primer extension analysis performed with B. pertussis RNA and a katA‐specific primer. A catalase‐deficient strain of B. pertussis, DD900, was constructed by gene replacement. DD900 was more sensitive to killing by 1 and 5 mM H2O2 than the parental strain, BP339. However, there was no difference in the ability of DD900 and BP339 to survive for 2h in human PMNs. This suggests that catalase plays no significant role in the survival of B. pertussis within PMNs
Molecular analysis of the biosynthesis pathway of the α‐2,8 polysialic acid capsule by Neisseria meningitidls serogroup B
Authors: Edwards Ulrike , Müller Astrid , Hammerschmidt Sven , Gerardy‐Schahn Rita , Frosch Matthias
Published: October 2006

The genes encoding all enzymes necessary for capsular polysaccharide biosynthesis in Neisseria meningitidis B are located on a 5 kb DNA fragment within the chromosomal cps gene cluster. Nucleotide sequence analysis revealed four open reading frames (ORFs), which can encode proteins with molecular masses of 41.4 kDa, 24.9kDa, 38.3 kDa, and 54.4 kDa, respectively. These ORFs constitute a transcriptional unit as demonstrated by Northern blots. Primer extension analysis revealed that the transcriptional start site is preceded by a nucleotide sequence with homologies to the σ70consensus promoter sequence of Escherichia coli. Functional analysis of the proteins encoded by the ORFs indicated that ORF2 encodes the CMP‐NeuNAc synthetase, ORF3 encodes the NeuNAc condensing enzyme, and ORF4 encodes the α‐2,8 polysialyltransferase. ORF1 encodes an enzyme, which provides a precursor molecule for synthesis of monomeric NeuNAc. In E. coli the subcloned ORFs 2–4 were able to synthesize a high‐molecular‐weight α‐2,8 polysialic acid. In contrast, inactivation of ORF1 in the meningococcal genome resulted in a complete loss of capsule production. A regulatory enzyme, the CMP‐NeuNAc hydrolase, which cleaves CMP‐NeuNAc to CMP and NeuNAc, was not found as a part of the capsular polysaccharide biosynthesis gene operon or within the cps gene cluster.
Distinct mechanisms of interactions of Opc‐expressing meningococci at apical and basolateral surfaces of human endothelial cells; the role of integrins in apical interactions
Authors: Virji Mumtaz , Makepeace Katherine , Moxon E. Richard
Published: October 2006

Interactions of Opc‐expressing Neisseria meningitidis with polarized and non‐polarized human umbilical vein endothelial cells (Huvecs) were investigated. Metabolic inhibitors and cytochalasin D treatment showed that host cellular and cytoskeletal functions were important for Opc‐expressing bacterial association with Huvecs at the apical surface. In addition, this interaction required the presence of serum in the incubation medium whilst association with nonpolarized cells did not require serum. Pre‐exposure of Opc‐expressing bacteria to serum was sufficient to increase the number of bacterial interactions at the apical surface; B306, a monoclonal antibody (mAb) against Opc, inhibited these interactions, suggesting that Ope binds to serum factor(s) and this in turn increases adherence to Huvecs. The receptors involved in this ‘sandwich’ adherence belong to the integrin family since the interaction was inhibited by peptides containing the amino acid sequence arginine‐glycine‐aspartic acid (RGD) and the tetrapeptide RGDS (but not the peptide RGES) was inhibitory. Non‐polarized cells appeared to expose receptors/sites that bound to Opc‐expressing bacteria directly, did not require serum factors and were not inhibited by RGD‐containing peptides. Serum‐dependent interactions of Opc‐expressing bacteria to apical surface was inhibited significantly by severai mAbs against avβ3 integrins. Some mAbs against α5 and β1 caused partial inhibition; antibodies that did not block the function of β1 integrins or the mAbs against α2 integrins were not inhibitory to bacterial interactions with Huvecs. Purified vitronectin supported adherence of Opc‐expressing bacteria to Huvecs but not of Opc‐bacteria. These interactions were inhibited by mAb B306 against Opc, by RGDS peptides as well as by blocking antibodies directed against αvβ‐3 but not antibodies against other integrins. These data suggest that a sequence of molecular events resulting in trimolecular complexes at the endothelial surface may drive neisserial invasion of Huvesc. The expression of Opc appears to enable bacteria to utilize the normal signal‐transduction mechanism of host cells via ligands in sera that adhere to endothelial cell integrins.
A Haemophilus influenzae IgA protease‐like protein promotes intimate interaction with human epithelial cells
Authors: St. Geme Joseph W. , Maria L. de la Morena  , Falkow Stanley
Published: October 2006

Haemophilus influenzae represents a common cause of human disease and an important source of morbidity and mortality. Disease caused by this organism begins with colonization of the upper respiratory tract. Several studies indicate that H. influenzae is capable of binding to and entering cultured human cells, properties which are potentially of relevance to the process of colonization. In the present study, we isolated an H. influenzae gene designated hap, which is associated with the capacity for In vitro attachment and entry. Analysis of the derived amino acid sequence of hap demonstrated significant homology with the serine‐type lgA1 proteases expressed by H. influenzae and Neisseria gonorrhoeae. It is notable that the hap product shares the catalytic domain of the lgA1 proteases and appears to be processed and secreted in an analogous manner. We speculate that the hap gene product is an important determinant of colonization, perhaps enabling the organism to evade the local immune response and thereby persist within the respiratory tract.
Growth and viability of Streptomyces coelicolor mutant for the cell division gene ftsZ
Authors: McCormick Joseph R. , Su Edwin P. , Driks Adam , Losick Richard
Published: October 2006

A homologue of the bacterial cell division gene ftsZ was cloned from the filamentous bacterium Streptomyces coelicolor. The gene was located on the physical map of the chromosome at about ‘11 o'clock’ (in the vicinity of glkA, hisA and trpB). Surprisingly, a null mutant in which the 399‐codon ftsZ open reading frame was largely deleted was viable, even though the mutant was blocked in septum formation. This indicates that cell division may not be essential for the growth and viability of S. coelicolor. The ftsZ mutant was able to produce aerial hyphae but was unable to produce spores, a finding consistent with the idea that ftsZ is required in order for aerial hyphae to undergo septation into the uninucleoid cells that differentiate into spores.
Observations on the formation of deletions on monomeric and dimeric plasmids in Escherichia coli
Authors: Ravn Peter , Givskov Michael , Eegholm Karen‐Magrethe , Madsen Søren M. , Boe Lars
Published: October 2006

We have studied the formation of spontaneous mutations on plasmids present In the monomeric and dimeric states in a recF strain of Escherichia coli. Two test systems were employed: (i) the precise excision of Tn5 from the tetA gene of the plasmid pBR322 and (ii) operator constitutive (Oc) mutations on the pBR322‐derived plasmid pPY97. The rate of Oc mutations was increased by a factor of three when this plasmid was present in the dimeric state compared to the monomeric state and the Oc phenotype was caused by small deletions in the operator sequence. No apparent mutational hot‐spot was found. The rate of Tn5 excision was increased on dimeric compared to monomeric plasmids. Excision from a dimeric plasmid usually resulted in two types of mutant plasmids; a dimeric plasmid, where the Tn5 had excised from one of the plasmid units, and a monomeric parental pBR322. A mechanism to account for this is suggested. Complementation tests revealed that the increased mutation rate on dimeric plasmids is the result of dimers being mutaphilic per se, rather than the result of a general, trans‐acting increase in mutation rates of the host, induced by the presence of the dimeric plasmid. Furthermore, it was found that the rate of Tn5 excision from plasmids in the monomeric state was increased when the region carrying the inserted Tn5 was duplicated.
Permeability of the cell wall of Mycobacterium smegmatis
Authors: Trias Joaquim , Benz Roland
Published: October 2006

The cell wail of Mycobacterium smegmatis me2155 was shown to be an effective permeability barrier to hydrophilic compounds. Permeability coefficients to β‐lactams ranged from 10 × 10 −7 to 0.5 × 10 −7 cm s−1. Cell wall proteins were solubilized with EDTA and Genapol and were tested for channel‐forming activity by reconstitution into lipid bilayers. Proteins were able to induce a voltage‐gated cation‐selective channel. The mycobacterial porin channel appeared to be water‐filled since the single‐channel conductance followed the mobility sequence of hydrated ions in the aqueous phase. On the basis of the Renkin equation and the single‐channel conductance, the channel diameter was estimated to be around 3 nm. Model calculations showed that cation selectivity may be caused by four negative point‐charges at the channel mouth. The permeability properties of the cell wall of intact cells were in good agreement with those of the reconstituted channel. Negatively charged cephalosporins, cefamandole and cephalothin, diffused at a 10‐ to 20‐fold lower rate than the zwitterionic cephaloridine. The mycobacterial porin represents a major hydrophilic pathway of the cell wall of M. smegmatis.
Overexpression of fIbA, an early regulator of Aspergillus asexual sporulation, leads to activation of brIA and premature initiation of development
Authors: Lee Bee Na , Adams Thomas H.
Published: October 2006

Aspergillus nidulans reproduces asexually by forming thousands of mitotically derived spores atop highly specialized multicellular organs termed conidiophores. We have identified a gene called flbA (for fluffy low brlA expression) that is required for initiation of A. nidulans conidiophore development. flbA mutants form abnormal colonies that have a distinct fluffy phenotype characterized by tightly interwoven aerial hyphae that autolyse as the colony matures. The requirement for fIbA in conidiophore development precedes activation of brlA, a primary regulator of conidiophore development. The wild‐type flbA gene was isolated and found to encode a 3.0 kb mRNA that is expressed throughout the A. nidulans asexual life cycle. Overexpression of fIbA using an Inducible promoter resulted in misscheduled expression of brlA in vegetative ceils and caused hyphal tips to differentiate into spore‐producing structures. Sequence analysis of a nearly full‐length fIbA cDNA clone showed that fibA is predicted to encode a 717‐amino‐acid polypeptide with 30% identity to the Saccharomyces cerevisiae SST2 protein. SST2 is required by yeast cells for resuming growth following prolonged exposure to yeast mating pheromone and for mating partner discrimination. We propose that fIbA plays a related role in a signalling pathway for Aspergillus conidiophore development.
Multiple protein‐DNA and protein‐protein interactions are involved in transcriptional activation by MaIT
Authors: Danot Olivier , Raibaud Olivier
Published: October 2006

The promoters of the Escherichia coli Maltose regulon are positively regulated by the MaIT protein, which recognizes a short asymmetric nucleotide sequence that is present as several copies in each promoter of the regulon. We report a detailed biochemical characterization of the interaction of MaIT with the promoter of the malPQ operon. The MaIT sites in malPp were precisely located and their occupation as a function of MaiT concentration was quantified using DNase I and dimethyl sulphate footprinting experiments. The contribution of each site to malPp activity was assessed by Introducing mutations that destroy them and measuring the residual in vivo and in vitro activity. Two main results were obtained. First, although the proximal MaIT site is centred at ‐37.5, RNA polymerase is likely to establish a contact required for matPp activity with at least one base pair of the promoter ‐35 region; this close proximity between RNA polymerase and MaIT bound to site 1 suggests that the two proteins interact. Second, even if the interaction of MaIT with the three functional sites in maIPp is a co‐operative process, the MaIT molecules bound to the two distal sites play a more subtle rote than simply increasing the occupancy of the proximal site and may also contact RNA polymerase. We suggest that, in the nucleoprotein structure responsible for the initiation of transcription, MaIT, RNA polymerase and malPp are held together by several weak interactions.
Klebsiella pneumoniae genes for citrate lyase and citrate lyase ligase: localization, sequencing, and expression
Authors: Bott Michael , Dimroth Peter
Published: October 2006

In the course of studies on anaerobic citrate metabolism in Klebsiella pneumoniae, the DNA region upstream of the gene for the sodium‐dependent citrate carrier (dtS) was investigated. Nucleotide sequence analysis revealed a cluster of five new genes that were oriented inversely to citS and probaby form an operon. The genes were named citCDEFG. Based on known protein sequence data, the gene products derived from citD, citE and citF could be identified as the λ‐, β‐, and α‐subunits of citrate lyase, respectively. This enzyme catalyses the cleavage of citrate to oxaloacetate and acetate. The gene product derived from citC (calculated Mr 36476) exhibited no obvious similarity to other proteins. In the presence of acetate and ATP, cell extracts from a citC‐expressing Escherichia coli strain were able to reactivate purified citrate lyase from K. pneumoniae that had been inactivated by chemical deacetylation of the prosthetic group. This represents 5‐phosphoribosyi‐dephospho‐acetyl‐coenzyme A which is covalently bound to serine‐14 of the acyl carrier protein (λ‐subunit). CitC was thus identified as acetate:SH‐citrate lyase ligase. The function of the gene product derived from citG (Mr 32 645) has not yet been identified. Expression of the CitCDEFG gene cluster in E. coli led to the formation of citrate lyase which was active only in the presence of acetyl‐coenzyme A, a compound known to substitute for the prosthetic group. These and other data strongly indicated that the enzyme synthesized in E. coli lacked its prosthetic group. Thus, additional genes besides citCDEFG appear to be required for the formation of holo‐citrate lyase.
Mutants at conserved positions in gene IV, a gene required for assembly and secretion of filamentous phages
Authors: Russel Marjorie
Published: October 2006

The filamentous phage protein pIV is required for assembly and secretion of the virus and possesses regions homologous to those found in a number of Gram‐negative bacterial proteins that are essential components of a widely distributed extracellular protein‐export system. These proteins form multimers that may constitute an outer membrane channel that allows phage/protein egress. Three sets of f1 gene IV mutants were isolated at positions that are absolutely (G355 and P375) or largely (F381) conserved amongst the 16 currently known family members. The G355 mutants were non‐functional, interfered with assembly of plV+ phage, and made Escherichia coli highly sensitive to deoxycholate. The P375 mutants were non‐functional and defective in multimerization. Many of the F381 mutants retained substantial function, and even those in which charged residues had been introduced supported some phage assembly. Some inferences about the roles of these conserved amino acids are made from the mutant phenotypes.
Significance of plasmolysis spaces as markers periseptal annuli and adhesion sites
Authors: Woldringh Conrad L.
Published: October 2006

During hyperosmotic shock, the protoplast and stretched‐out peptidoglycan layer first shrink together until the turgor pressure in the cell is relieved. Being non‐compressible, the outer and inner membranes must fold their superfluous surfaces. While the protoplast contracts further, the inner membrane rearranges into plasmolysis spaces visible by phase contrast microscopy. Two opposing theories predict a similar positioning of spaces in dividing cells and filaments: the ‘periseptal annulus model, based on adhesion zones, involved in the predetermination of the division site; and a ‘restricted, random model’, based on physical properties of the protoplast. Strong osmotic shock causes retraction of the inner membrane over almost the entire surface forming the so‐called ‘Bayer bridges’. These tubular adhesion sites are preserved by chemical fixation, and can be destroyed by cryofixation and freeze‐substitution of unfixed ceils. Both the regular positioning of the plasmolysis spaces and the occurrence of tubular adhesion sites can be explained on the basis of physical properties of the membrane which necessitate rearrangements by membrane flow during shrinkage of the protoplast.
Lipooligosaccharide biosynthesis in Neiseria gonorrhoeae: cloning, identification and characterization of the α1,5 heptosyltransferase I gene (rfaC)
Authors: Zhou Daoguo , Lee Na‐Gyong , Apicella Michael A.
Published: October 2006

The identical partial deep‐core structure of Hepα1–3Hepα1–5KDO In Salmonella typhimurium LT2 LPS and Neisseria gonorrhoeae LOS enabled us to isolate a DNA fragment from N. gonorrhoeae that was able to complement the α1,5 LOS heptosyltransferase defect in the S. typhimurium rfaC630 (SA1377) mutant. SDS‐PAGE analysis confirmed the production of wild‐type LPS in the transformant. Subcloning revealed that complementation was due to a 1.2 kb fragment. Sequence analysis revealed a complete open reading frame capable of encoding a 36–37 kDa peptide. In vitro transcription‐translation analysis of the 1.2 kb clone confirmed that a 37 kDa protein was encoded by this DNA fragment. The DNA sequence‐deduced protein had 36% identity and 58% similarity to S. typhimurium heptosyltransferase I (RfaC). Primer extension analysis indicated that transcription of the cloned gene in N. gonorrhoeae strain 1291 begins 144bp upstream of the start codon at a G nucleotide. An isogenic mutant of N. gonorrhoeae strain 1291 with an m‐Tn3 insertion inside the coding sequence expressed a single truncated LOS with a similar molecular mass to S. typhimurium rfaC LPS. We conclude that the 1.2 kb fragment encodes the α1,5 LOS heptosyltransferase 1 (RfaC) in N. gonorrhoeae. Our studies also provide further evidence that the third KDO residue in S. typhimurium LPS is added after the core synthesis is completed.
Purification of a MaIE‐SoxS fusion protein and identification of the control sites of Escherichia coli superoxide‐inducible genes
Authors: Fawcett William P. , Wolf Richard E.
Published: October 2006

In Escherichia coli, the soxRS genes effect the cell's defence against superoxide by activating the transcription of more than 14 genes, including zwf, sodA, nfo, micF and fumC. Previous work from other laboratories has Indicated that SoxR is the sensor of oxidative stress and induces synthesis of SoxS, which in turn activates transcription of the regulon's target genes. Although SoxS appears to be a DNA‐binding protein, its ability to bind to the promoter regions of target genes has not been demonstrated. To facilitate purification and characterization of SoxS, we constructed a fusion of soxS to MalE, which encodes maltose‐binding protein, and demonstrated that the in vivo expression of the MaIE‐SoxS fusion protein can provide SoxS function to a soxRS deletion mutant. We purified the fusion protein by affinity chromatography on an amylose column. The purified fusion protein stimulated m vitro expression of zwf in a coupled transcription‐translation system and formed specific complexes with DNA fragments carrying target gene promoters. Moreover, MalE–SoxS protected from DNase I attack 22–27 bp segments immediately adjacent to or overlapping the −35 hexamers of the zwf, sodA, nfo, micF. and fumC promoters. The protected regions revealed a consensus ‘soxbox’ sequence.
Characterization and occurrence of two repeated palindromic DNA elements of Brucella spp.: Bru‐RS1 and Bru‐RS2
Authors: Halling Shirley M. , Bricker Betsy J.
Published: October 2006

Two repeated DNA elements of 103 bp and 105 bp were discovered in brucellae and designated Bru‐RS1 and Bru‐RS2, respectively. The two elements are palindromic, are 65% similar in sequence, form two families of elements that are slightly divergent in sequence, appear to be intergenic, and are found, collectively, in more than 35 copies in brucellae. These elements are bounded by perfect or nearly perfect inverted repeats. A third copy of the terminal repeat is found within the elements and is the terminus for several truncated copies of the Bru‐RS1 family. Hybridization patterns for the elements among brucellae were unique. The elements are dispersed, highly conserved among brucellae, and hot‐spots for insertion by IS711.
Non‐motile mutants of Helicobacter pylori and Helicobacter mustelae defective in flagellar hook production
Authors: O'Toole Paul W. , Kostrzynska Magdalena , Trust Trevor J.
Published: October 2006

Flagellar hooks were purified from Helicobacter pylori and Helicobacter mustelae. The 70 × 16nm H. pylori hook was composed of FIgE subunits of 78kDa, while the 72 × 16nm H. mustelae hook was composed of 87kDa subunits. N‐terminal sequence was obtained for the FIgH proteins of both species, and for an internal H. mustelae FlgE peptide. Degenerate oligonucleotide primers allowed amplification of a 1.2 kb fragment from the H. mustelae chromosome, which carried part of the flgE gene. The corresponding H. pylori gene was cloned by immunoscreening of a genomic library constructed in λZAP Express, The translated H. pylori flgE sequence indicated a protein with limited homology with the hook proteins from Salmonella typhimurium and Treponema phagedenis. Mutants of H. pylori and H. mustelae defective in hook production generated by allele replacement were non‐motile and devoid of flagellar filaments but produced both flagellin subunits, which were localized in the soluble fraction of the cell. The level of flagellin production was unchanged in the mutants, indicating that the regulation of flagellin expression in Helicobacter differs from that in the Enterobacteriaceae.
The ccoNOQP gene cluster codes for a cb‐type cytochrome oxidase that functions in aerobic respiration of Rhodobacter capsulatus
Authors: Thöny‐Meyer Linda , Beck Christoph , Preisig Oliver , Hennecke Hauke
Published: October 2006

The genes for a new type of a haem‐copper cytochrome oxidase were cloned from Rhodobacter capsulatus strain 37b4, using the Bradyrhizobium japonicum fixNOQP gene region as a hybridizing probe. Four genes, probably organized in an operon (ccoNOQP), were identified; their products share extensive amino acid sequence similarity with the FixN, O, Q and P proteins that have recently been shown to be the subunits of a cb‐type oxidase. CcoN is a c‐type cytochrome, CcoO and CcoP are membrane‐bound mono‐ and dihaem c‐type cytochromes and CcoQ is a small membrane protein of unknown function. Genes for a similar oxidase are also present in other non‐rhizobial bacterial species such as Azoto‐bacter vinelandii, Agrobacterium tumefaciens and Pseudomonas aeruginosa, as revealed by polymerase chain reaction analysis. A ccoN mutant was constructed whose phenotype, in combination with the structural information on the gene products, provides evidence that the CcoNOQP oxidase is a cytochrome c oxidase of the cb type, which supports aerobic respiration in R. capsulatus and which is probably identical to the cbb3‐type oxidase that was recently purified from a different strain of the same species. Mutant analysis also showed that this oxidase has no influence on photosynthetic growth and nitrogen‐fixation activity.
mRNA degradation in Escherichia coli: a novel factor which impedes the exoribonucleolytic activity of PNPase at stem‐loop structures
Authors: Causton Helen , Py Béatrice , McLaren Robert S. , Higgins Christopher F.
Published: October 2006

Stem‐loop structures can protect upstream mRNA from degradation by impeding the processive activities of 3′–5′ exoribonucleases. The ability of such structures to impede exonuclease activity in vitro is insufficient to account for the stability they can confer on mRNA in vivo. In this study we identify a factor from Escherichia coli which specifically impedes the processive activity of the 3′–5′ exonuclease PNPase at stem‐loop structures in vitro. This factor can, potentialiy, reconcile the apparent discrepancy between the ability of 3′ stem‐loop structures to stabilize upstream mRNA in vitro and in vivo. Its mechanism of action, and possible role in regulating mRNA degradation, is discussed.
Transmembrane signalling by the chimeric chemosensory receptors of Escherichia coli Tsr and Tar with heterologous membrane‐spanning regions
Authors: Tatsuno Ichiro , Lee Lan , Kawagishi Ikuro , Homma Michio , Imae Yasuo
Published: October 2006

The serine and aspartate chemosensory receptors (Tsr and Tar) of Escherichia coli have two membrane‐spanning regions TM1 and TM2. To investigate their roles in transmembrane signalling, we constructed two chimeric receptors from Tsr and Tar with heterologous combinations of TM1 and TM2: the N‐terminus of one receptor, including TM1 and the periplasmic domain, was fused to the C‐terminus of the other, beginning with TM2. Both of the chimeric receptor genes rescued the chemotactic defect of a receptorless E. coli strain, indicating that the chimeric receptors are functional. Their apparent affinities for the specific ligands were the same as those of Tsr or Tar. Therefore, as far as transmembrane signalling abilities are concerned, the TW2 regions of Tsr and Tar are interchangeable, suggesting that sequence‐specific interaction between TM1 and TM2 may not be required for the signal transmission across the membrane.

The cells expressing either of the chimeric receptors, however, showed ‘smooth’, biased, basal swimming patterns. Moreover, they adapted quickly after stimulation with the repellent glycerol. This rapid adaptation was observed even in the methyltransferase‐defective strain. Therefore, exchange of TM2 might impose structural constraints on the chimeric receptors that stabilize conformations which elicit smooth swimming.
SeqA limits DnaA activity in replication from oriC in Escherichia coli
Authors: Ulrik von Freiesleben  , Rasmussen Knud V. , Schaechter Moselio
Published: October 2006

A mutant Escherichia coli that transforms minichromosomes with high efficiency in the absence of Dam methylation has been Isolated and the mutation mapped to 16.25 min on the E. coli map. The mutant strain containing seqA2 is defective for growth in rich medium but not in minimal medium. A similar mutation In this gene, named seqA1, has also been isolated.

Here we show that the product of the seqA gene, SeqA, normally acts as an inhibitor of chromosomal initiation. In the seqA2‐containing mutant, the frequency of initiation increases by a factor of three. Introduction of the wild‐type seqA gene on a low‐copy plasmid suppresses the cold sensitivity of a dnaAcos mutant known to overinitiate at temperatures below 39°C. In addition, the seqA2 mutation is a suppressor of several dnaA (Ts) alleles.

The seqA2 mutant overinitiates replication from oriC and displays the asynchronous initiation phenotype. Also the seqA2 mutant has an elevated level of DnaA protein (twofold).

The introduction of minichromosomes or a low‐copy‐number plasmid carrying five DnaA‐boxes from the oriC region increases the growth rate of the seqA2 mutant in rich medium to the wild‐type level, reduces overinitiation but does not restore synchrony. We propose that the role of SeqA is to limit the activity level of the E. coli regulator of chromosome initiation, DnaA.
Multiple roles for DNA polymerase I in establishment and replication of the promiscuous plasmid pLS1
Authors: Díaz Asunción , Lacks Sanford A. , López Paloma
Published: October 2006

The polymerase activity of DNA polymerase I is important for the establishment of the pLS1 replicon by reconstitutive assembly in Streptococcus pneumoniae after uptake of exogenous pLS1 plasmid DNA. In polA mutants lacking the polymerase domain, such establishment was reduced at least 10‐fold in frequency. Chromosomally facilitated establishment of pLS1‐based plasmids carrying DNA homologous to the host chromosome was not so affected. However, both types of plasmid transfer gave mostly small colonies on initial selection, which was indicative of a defect in replication and filling of the plasmid pool. Once established, the pLS1‐based plasmids replicated in polA mutants, but they showed segregational instability. This defect was not observed in strains with the wild‐type enzyme or in an S. pneumoniae strain that encodes the polymerase and exonuclease domains of the enzyme on separate fragments. The role of DNA polymerase I in stably maintaining the plasmids depends on its polymerizing function in three separate steps of rolling‐circle replication, as indicated by the accumulation of different replication intermediate forms in polA mutants. Furthermore, examination of the segregational stability of the pLS1 replicon in an Escherichia coli mutant system indicated that both the polymerase and the 5′‐to‐3′ exonuclease activities of DNA polymerase I function in plasmid replication.
Genes required for both gliding motility and development in Myxococcus xanthus
Authors: MacNeil Spencer D. , Mouzeyan Aram , Hartzell Patricia L
Published: October 2006

Myxococous xanthus cells can glide both as individual cells, dependent on Adventurous motility (A motility), and as groups of cells, dependent upon Social motility (S motility), Tn5‐lac mutagenesis was used to generate 16 new A‐ and nine new S‐ mutations. In contrast with previous results, we find that subsets of A‐ mutants are defective in fruiting body morphogenesis and/or myxospore differentiation. All S‐ mutants are defective in fruiting body morphogenesis, consistent with previous results. Whereas some S‐ mutants produce a wild‐type complement of spores, others are defective in the differentiation of myxospores. Therefore, a subset of the A genes and all of the S genes are critical for fruiting body morphogenesis. Subsets of both A and S genes are essential for sporulation. Three S::Tn5–lac insertions result in surprising phenotypes. Colonies of two S‐ mutants glide on ‘swim’ (0.35% agar) plates to form fractal patterns. These S‐ mutants are the first examples of a bacterium in which mutations result in fractal patterns of colonial spreading. An otherwise wild‐type strain with one S‐ insertion resembles the frz‐ sglA1‐ mutants upon development, suggesting that this S‐ gene defines a new chemotaxis component in M. xanthus.
Requirement of the regulatory protein NtcA for the expression of nitrogen assimilation and heterocyst development genes in the cyanobacterium Anabaena sp. PCC7120
Authors: Frías José E. , Flores Enrique , Herrero Antonia
Published: October 2006

The cyanobacterial ntcA gene encodes a DNA‐binding protein that belongs to the Crp family of bacterial transcriptional regulators. In this work, we describe the isolation of an ntcA insertional mutant of the dinitrogen‐fixing, heterocyst‐forming cyanobacterium Anabaena sp. PCC 7120. The Anabaena ntcA mutant was able to use ammonium as a source of nitrogen for growth, but was unable to assimilate atmospheric nitrogen (dinitrogen) or nitrate. Nitrogenase and enzymes of the nitrate reduction system were not synthesized in the ntcA mutant under derepressing conditions, and glutamine synthetase levels were lower in the mutant than in the wild‐type strain. In the ntcA mutant, in response to removal of ammonium, accumulation of mRNA of the genes encoding nitrogenase (nifHDK), nitrite reductase (nir, the first gene of the nitrate assimilation operon), and glutamine synthetase (glnA) was not observed. A transcription start point of the Anabaena glnA gene (corresponding to RNA1), that has been shown to be used preferentially after nitrogen step‐down, was not used in the ntcA insertional mutant. Heterocyst development (which is necessary for the aerobic fixation of dinitrogen) and induction of hetR (a regulatory gene that is required for heterocyst development) were also impaired in the ntcA mutant. These results showed that the ntcA gene product, NtcA, is required in Anabaena sp. PCC 7120 for the expression of genes encoding proteins involved in the assimilation of nitrogen sources alternative to ammonium including dinitrogen and nitrate, and that the process of heterocyst development is also controlled by NtcA.
Transcriptional attenuation control of the tylosin‐resistance gene tlrA in Streptomyces fradiae
Authors: Kelemen Gabriella H. , Zalacain Magdalena , Culebras Esther , Seno Eugene T. , Cundliffe Eric
Published: October 2006

The tylosin producer Streptomyces fradiae contains four known resistance genes, two of which (tlrA and tlrD) encode methyltransferases that act on ribosomal RNA at a common site. Expression of tlrA is regulated via transcriptional attenuation. A short transcript, only 411 nucleotides long, terminates 27 nucleotides into the methylase‐coding sequence in the uninduced state. Induction of tlrA is proposed to involve a ribosome‐mediated conformational change within the mRNA leader that allows transcription to continue beyond the attenuation site, resulting in a transcript about 1450 nucleotides long. Transplantation of tlrD and/or tlrA into Streptomyces albus revealed that the induction specificity of tlrA depends upon the state of the ribosomes and is significantly altered in strains also expressing tlrD.
Iron piracy: acquisition of transferrin‐bound iron by bacterial pathogens
Authors: Cornelissen Cynthia Nau , Sparling P. Frederick
Published: October 2006

The mechanism of iron utilization from transferrin has been most extensively characterized in the pathogenic Neisseria species and Haemophilus species. Two transferrin‐binding proteins, Tbp1 and Tbp2, have been identified in these pathogens and are thought to be components of the transferrin receptor. Tbp1 appears to be an integral, TonB‐dependent outer membrane protein while Tbp2, a lipoprotein, may be peripherally associated with the outer membrane. The relative contribution of each of these proteins to transferrin binding and utilization is discussed and a model of iron uptake from transferrin is presented. Sequence comparisons of the genes encoding neisserial transferrin‐binding proteins suggest that they are probably under positive selection for variation and may have resulted from inter‐species genetic exchange.
Specificity domain localization of Bacillus thuringiensis insecticidal toxins is highly dependent on the bioassay system
Authors: Masson Luke , Mazza Alberto , Gringorten Larry , Baines Danica , Aneliunas Victoria , Brousseau Roland
Published: October 2006

The Bacillus thuringiensis cryIA(a) and cryIA(c) gene specificity regions were probed by creating and testing hybrid toxins both in vivo and in vitro against cultured insect cells or dissociated midgut epithelial cells. Toxin threshold dose determinations revealed that CryIA(c) is highly active against cultured Choristoneure fumiterana cells (CF‐1) whereas CryIA(a) is nontoxic. In live insect bioassays, a reversed order of toxicity was observed. Hybrid analysis reversed that the CryIA(c) toxicity‐determining region is located between codons 258 and 510. Two smaller subsections of this region (residues 258–358 and 450–510) were able to confer toxicity, although at lower levels, and one region (358–450) was present where progressive substitutions of CryIA(a) with cryIA(c) sequences had no effect. Exchanging the non‐homologous N‐terminal regions of CryIA(c) with CryIE suggested that the W‐terminus does not play a role in specificity. One hybrid clone, MP80, displays a 99.3% homology to CryIA(b) but shows an 800‐fold increase in toxicity to CF–1 cells relative to that shown by CryIA(b). Direct comparison between live Bombyx mori bioassays and a newly developed in vitro lawn assay using dissociated midgut epithelial cells from the same insect revealed striking differences in toxicity. The toxicity‐determining region for B. mori larvae was determined to be between codons 283 and 450, although the 450–620 codon region may exert an influence on toxicity. In general, native or hybrid toxins showing little or no insect intoxication were very active against the epithelial cells, suggesting that factors other than toxin amino acid sequence play an important role in determining toxin specificity.
Successive action of Escherichia coli chaperones in vivo
Authors: Gaitanaris George A. , Vysokanov Alexander , Hung Siu‐Chun , Gottesman Max E , Gragerov Alexander
Published: October 2006

Escherichia coli DnaK, DnaJ and GrpE are required for renaturation of heat‐inactivated λ CI857 repressor (Gaitanaris et al., 1990). Here we demonstrate that in addition to the above three proteins, GroEL and GroES are necessary for the CI857 repressor to acquire full activity at the permissive temperature. Although full‐length soluble repressor is present at normal amounts, the protein has reduced specific activity and migrates abnormally on native gels. To determine where the different chaperones act in protein folding, we identified their cellular locations. DnaK and DnaJ are associated with nascent polypeptide chains in translating ribosomes. In contrast, GroEL, although it is transiently associated with newly synthesized proteins, is absent from the ribosomes. This suggests that DnaK and DnaJ ptay an early role in protein maturation, whereas GroEL acts at a later stage.
Topological and mutational analysis of KpsM, the hydrophobic component of the ABC‐transporter involved in the export of polysialic acid in Escherichia coli K1
Authors: Pigeon Ronald P. , Silver Richard P.
Published: October 2006

The 17 kb kps gene cluster of Escherichia coli K1, which encodes the information required for synthesis, assembly and translocation of the polysialic acid capsule of E. coli K1, is divided into three functional regions. Region 3 contains two genes, kpsM and kpsT, essential for the transport of capsule polymer across the cytoplasmic membrane. The hydrophobicity profile of KpsM suggests that it is an integral membrane protein while KpsT contains a consensus ATP‐binding site. KpsM and KpsT belong to the ATP‐binding cassette (ABC) superfamily of membrane transporters. In this study, we investigate the topology of KpsM within the cytoplasmic membrane using β‐lactamase fusions and alkaline phosphatase sandwich fusions. Our analysis provides evidence for a model of KpsM having six membrane‐spanning regions, with the N‐ and C‐terminal domains facing the cytoplasm, and a short domain within the third periplasmic loop, which we refer to as the SV–SVI linker localizing in the membrane. Protease digestion studies are consistent with regions of KpsM exposed to the periplasmic space. In vivo cross‐linking studies provide support for dimerization of KpsM within the cytoplasmic membrane. Linker‐insertion and site‐directed mutagenesis define the N‐terminus, the first cytoplasmic loop, and the SV‐SVI linker as regions that are important for the function of KpsM in K1 polymer transport.
Regulation of the Yersinia enterocolitica enterotoxin Yst gene. Influence of growth phase, temperature, osmolarity, pH and bacterial host factors
Authors: Mikulskis Alvydas V. , Delor Isabelle , Thi Vinh Ha , Cornelis Guy R.
Published: October 2006

The chromosome of Yersinia enterocolitica encodes an enterotoxin called Yst. We analysed transcription of chromosomal yst′–luxAB and plasmid‐borne yst′–lacZ operon fusions and we observed that regulation of yst expression occurs at transcriptional level. In a wild‐type strain, yst was transcribed from at least two major promoters, yst transcription reached a maximum at the entry to the stationary phase and significantly varied in different Y. enterocolitica strains. In some strains, it gradually decreased during the course of our work, suggesting the existence of a mechanism switching the expression of yst to a silent state. Changes in the status of bacterial host factors rather than modifications in the yst gene are responsible for this silencing. Negative regulator YmoA participates in yst silencing and temperature regulation of yst YmoA was also required for proper growth‐phase regulation of yst, although it is not the only factor involved in this regulation. Physicochemical parameters of the environment play an important role in yst transcription. In usual culture media (e.g. tryptic soy broth), the enterotoxin gene was transcribed only at temperatures below 30°C, which argued against the role of Yst in a prolonged diarrhoea at body temperatures. However, yst transcription could be induced at 37°C by increasing osmolarity and pH to the values normally present in the ileum lumen. This finding reconciles the observations concerning yst expression in a host environment and in bacterial cultures, thus supporting the idea that enterotoxin Yst is a virulence factor of Y. enterocolitica.
Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus
Authors: Jaton‐Ogay Katia , Paris Sophie , Huerre Michel , Quadroni Manfredo , Falchetto Rocco , Togni Giuseppe , Latgé Jean‐Paul , Monod Michel
Published: October 2006

Aspergillus fumigatus secretes a serine alkaline protease (ALP) and a metalloprotease (MEP) when the fungus is cultivated in the presence of collagen as sole nitrogen and carbon source. The gene encoding ALP was isolated and characterized previously. We report here the cloning and the sequencing of the gene encoding MEP. Genomic and cDNA clones were isolated from A. fumigatus libraries using synthetic oligonucleotides as probes. Stretches of the deduced amino acid sequence were found to be in agreement with the N‐terminal amino acid sequence of MEP and with internal peptide sequences. The amino acid sequence of the enzyme contains a putative active‐site sequence HEYTH homologous to the active site of other bacterial and eukaryotic zinc metalloproteases. Sequence analysis reveals that MEP has a pre‐proregion consisting of 245 amino acid residues preceding the 388 amino acid residues of the mature region (molecular mass of 42 kDa). An alp mep mutant, deficient in proteolytic activity at neutral pH in vitro, was constructed and tested for pathogenicity in a murine model. No difference in pathogenicity was observed between the wild‐type strain and the alp mep double mutant, suggesting that ALP and MEP are not essential for the invasion of the lung tissues by A. fumigatus.
Characterization of a gene responsible for the Na+/H+ antiporter system of alkalophilic Bacillus species strain C‐125
Authors: Hamamoto Tetsuo , Hashimoto Michizane , Hino Motohiro , Kitada Makio , Seto Yasuyuki , Kudo Toshiaki , Horikoshi Koki
Published: October 2006

An alkali‐sensitive mutant, 38154, of the alkalophilic Bacillus sp. strain C‐125 could not grow at an alkaline pH. The nucleotide sequence of a 3.7 kb parental DNA fragment that recovers the growth of 38154 at alkaline pH has four open reading frames (ORF1–4). By sub‐cloning the fragment, we demonstrated that a 0.25 kb DNA region is responsible for the recovery. Direct sequencing of the mutant's corresponding region revealed a G to A substitution. The mutation resulted in an amino acid substitution from Gly‐393 to Arg of the putative 0RF1 product, which was deduced to be an 804‐amino‐acid polypeptide with a molecular weight of 89 070. The N‐terminal part of the putative ORF1 product showed amino acid similarity to those of the chain‐5 products of eukaryotic NADH quinine oxidoreductases. Membrane vesicles prepared from 38154 did not show membrane potential (δψ)‐driven Na+/H+ antiporter activity. Antiporter activity was resumed by introducing a parental DNA fragment which recovered the mutant's alkalophily. These results indicate that the mutation in 38154 affects, either directly or indirectly, the electrogenic Na+/H+ antiporter activity. This is the first report which shows that a gene responsible for the Na+/H+ anti‐porter system is important in the alkalophily of alkalo‐philic microorganisms.
Ribosomal protein methylation in Escherichia coli: the gene prmA, encoding the ribosomal protein L11 methyltransferase, is dispensable
Authors: Vanet Anne , Plumbridge Jacqueline A. , Guérin Marie‐France , Alix Jean‐Hervé
Published: October 2006

The prmA gene, located at 72 min on the Escherichia coli chromosome, is the genetic determinant of ribosomal protein L11‐methyltransferase activity. Mutations at this locus, prmA1 and prmA3, result in a severely undermethylated form of L11. No effect, other than the lack of methyl groups on L11, has been ascribed to these mutations. DNA sequence analysis of the mutant alleles prmA1 and prmA3 detected point mutations near the C‐terminus of the protein and plasmids overproducing the wild‐type and the two mutant proteins have been constructed.

The wild‐type PrmA protein could be crosslinked to its radiolabelled substrate, S‐adenosyl‐L‐methionine (SAM), by u.v. irradiation indicating that it is the gene for the methyltransferase rather than a regulatory protein. One of the mutant proteins, PrmA3, was also weakly crosslinked to SAM. Both mutant enzymes when expressed from the overproducing plasmids were capable of catalysing the incorporation of 3H‐labelled methyl groups from SAM to L11 in vitro. This confirmed the observation that the mutant proteins possess significant residual activity which could account for their lack of growth phenotype. However, a strain carrying an in vitro‐constructed null mutation of the prmA gene, transferred to the E. coli chromosome by homologous recombination, was perfectly viable.
The site‐specific recombination system regulating expression of the Type 1 fimbrial subunit gene of Escherichia coli is sensitive to changes in DNA supercoiling
Authors: Dove Simon L. , Dorman Charles J.
Published: October 2006

We have studied the effect of altering the in vivo level of DNA supercoiling on the phase‐variable expression of the Escherichia coli fimA gene. Transcription from the fimA promoter was unaffected by changes in DNA supercoiling whether caused by the introduction of a topA::7n10 mutation or by inhibition of DNA gyrase with the antibiotic novobiocin. However, inversion of the fimA promoter fragment was altered in response to perturbation of DNA supercoiling. Specifically, inactivation of topA reduced the rate of promoter fragment inversion in both the ON‐to‐OFF and the OFF‐to‐ON directions. This effect correlated with the loss of functional topA and not with the global level of DNA supercoiling. Inhibition of DNA gyrase introduced a bias in favour of the OFF‐to‐ON inversion; the ON‐to‐OFF inversion was affected only slightly. Changes in expression of fimB, the gene coding for the recombinase that catalyses fimA promoter fragment inversion in the strains used In this study, did not correlate with effects on fimA phase variation: we found that transcription of fimB was inhibited by loss of functional topA and was enhanced by inhibition of DNA gyrase in a manner that correlated well with the global level of in vivo DNA supercoiling. A model is presented to account for the effects of lost topoisomerase function on fimA gene expression.