bluuebunny/crossref_metadata_2025 huggingface dataset:
PhoP/PhoQ: macrophage‐specific modulators of Salmonella virulence?
Authors: Miller S. I.
Published: October 2006

The regulation of gene expression by the two‐component regulatory system PhoP/PhoQ is necessary for Salmonella typhimurium survival within macro‐phages, defensin resistance, acid resistance, and murine typhoid fever pathogenesis. Salmonella experience multiple environments during mammalian infection and survival requires tightly regulated gene expression. Alter phagocytosis by macrophages, signal transduction by PhoQ results in the transcription of QhoP‐activated genes (pags) encoding proteins essential to bacterial survival and virulence. One such gene, pagC, encodes an envelope protein with amino acid similarity to an epithelial cell invasion protein of Yersina enterocolitica, Ail, and a bacteriophage λ outer membrane protein, Lom. The PhoP and PhoQ proteins can also repress the synthesis of proteins, encoded by phoP repressed genes (prgs), when pags are maximally expressed. If prgs encode receptors for toxic compounds, prg repression may protect the cell within macrophages when pag expression is most necessary. At least one prg locus, prgH, is required for full S. typhimurium mouse virulence. Within the macrophage, different environments may stimulate a switch from pag to prg expression that is necessary to Salmonella survival, prg expression may also be necessary for surviving non‐macrophage environments. Study of the PhoP regulon should lead to the discovery of new virulence factors, increase knowledge of how gene regulation is essential to bacterial virulence, and perhaps lead to the development of better vaccines for typhoid fever.

Salmonella species experience multiple environments within mammalian hosts
Energy requirements for protein translocation across the Escherichia coli inner membrane
Authors: Geller B. L.
Published: October 2006

Both ATP and an electrochemical potential play roles in translocating proteins across the Inner membrane of Escherichia coli. Recent discoveries have dissected the overall transmembrane movement into separate subreactions with different energy requirements, identified a translocation ATPase, and reconstituted both energy‐requiring steps of the reaction from purified components. A more refined understanding of the energetics of this fundamental process is beginning to provide answers about the basic issues of how proteins move across the hydrophobic membrane barrier.
Sequencing, mutational analysis, and transcriptional regulation of the Escherichia coli htrB gene
Authors: Karow M. , Georgopoulos C.
Published: October 2006

The Escherichia coli htrB gene was originally discovered because its insertional inactivation led to an exquisitely temperature‐sensitive phenotype in rich media, i.e. the ability to form colonies at temperatures below 32°C, but not above 33°C. The htrB gene has been sequenced. It can potentially code for two proteins, with Mr values of 35407 Da and 8669 Da, that are encoded by overlapping, divergent open reading frames. Our data are consistent with the 35407 Da protein being HtrB. Northern blot analysis clearly shows that the monocistronic htrB message is not under heat‐shock regulation. We have also sequenced the flanking DNA and have discovered a new gene, designated orf39.9, located immediately adjacent to htrB, but divergently transcribed.
On the evolution of functional secondary metabolites (natural products)
Authors: Stone M. J. , Williams D. H.
Published: October 2006

It is argued that organisms have evolved the ability to biosynthesize secondary metabolites (natural products) because of the selectional advantages they obtain as a result of the functions of the compounds. The clustering together of antibiotic biosynthesis, regulation, and resistance genes implies that these genes have been selected as a group and that the antibiotics function in antagonistic capacities in nature. Pleiotropic switching, the simultaneous expression of sporulation and antibiotic biosynthesis genes, is interpreted in terms of the defence roles of antibiotics. We suggest a general mechanism for the evolution of secondary metabolite biosynthesis pathways, and argue against the hypothesis that modern antibiotics had prebiotic effector functions, on the basis that it does not account for modern bio‐synthetic pathways.
spbA locus ensures the segregational stability of pTH1030, a novel type of Gram‐positive replicon
Authors: Lereclus D. , Arantes O.
Published: October 2006

The replication region of the plasmid pHT1030 of Bacillus thuringiensis was previously mapped to a 2.9 kb DNA fragment. The DNA sequence was analysed and it was shown that the minimal replicon resides within a 1 kb fragment of DNA carrying no potential protein coding sequence. Moreover, no production of single‐stranded DNA intermediates was detected in the plasmid‐containing cells. pHT1030 therefore belongs to a class of replicons not previously described in Gram‐positive bacteria. Examination of the segregational stability of deletion derivatives of pHT1030 in bacilli defined two stability regions. One is located within the minimal replicon of pHT1030, whereas the second (spbA) is not required for replication. spbA encodes a 15 kDa protein and ensures the segregational stability of the plasmid. This effect of spbA is particularly highlighted in sporulation. The absence of the spbA locus gives rise to plasmid‐free spores at high frequency, whereas the spbA+ plasmids are stably maintained. The stability of the plasmids during sporulation seems to be correlated with an unequal division of the cell by the sporulation septum.
Haemolysin‐derived synthetic peptides with pore‐forming and haemolytic activity
Authors: Oropeza‐Wekerle R.‐L. , Muller S. , Briand J.‐P. , Benz R. , Schmid A. , Goebel W.
Published: October 2006

Escherichia coli haemolysin (Hlya) is a pore‐forming protein which belongs to the family of ‘Repeat‐toxins’ (RTX)(Lo et al., 1989; Lally et al., 1989; Kraig et al., (1990). A model for the pore‐forming structure of HlyA has been proposed (Ludwig et al., 1991) which consists of eight transmembrane segments all present in this hydrophobic region of HlyA. We report here that two synthetic peptides of 10 and 8 amino acids in length (Pep1 and Pep2, respectively), which are derived from transmembrane segment V, are able to form pores in an artificial lipid bilayer. In addition, Pep1 exhibits strong haemolytic activity when tested on human red blood cells (HRBCs). The haemolytic activity of Pep1 and of E. coli haemolysin is completely inhibited by antibodies raised against Pep1.
Membrane‐associated GTPases in bacteria
Authors: March Paul E.
Published: October 2006

Members of the GTPase superfamily are extremely important in regulating membrane signalling pathways in all cells. This review focuses on membrane‐associated GTPases that have been described in prokaryotes. In bacteria, LepA and NodO are very similar to protein synthesis elongation factors but apparently have membrane‐related functions. The amino acid sequences of FtsY and Ffh are clearly related to eukaryotic factors involved in protein secretion. Obg and Era are not closely related to any GTPase subgroup according to amino acid sequence comparisons, but they are essential for viability. In spite of similarities to well‐studied eukaryotic proteins the signalling pathways of these cellular regulators, with the exception of NodQ, have not yet been elucidated.
A system of transposon mutagenesis for bacteriophage T4
Authors: Woodworth Denise L. , Kreuzer Kenneth N.
Published: October 2006

We have developed a system of transposon mutagenesis for bacteriophage T4. The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24, permitting a direct selection for transposition events into a gene 24‐deteted phage. The transposition occurred at a frequency of only 10‐7 per progeny phage, even though a dam‐ host was used to increase transposition frequency. Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon‐specific probe. Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions. The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome. In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred. Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.
The Aeromonas hydrophila exeE gene, required both for protein secretion and normal outer membrane biogenesis, is a member of a general secretion pathway
Authors: Jiang Bo , Howard S. Peter
Published: October 2006

The Aeromonas hydrophila Tn5–751 insertion mutant L1.97 is unable to secrete extracellular proteins, and is fragile because of defective assembly of its outer membrane. A Kpnl 4.1 kb fragment, which complements this mutant when supplied with an exogenous promoter, was isolated and sequenced. It contains two complete genes, exeE and exeF, plus fragments of two others and may form part of an operon. The exeE and exef open reading frames encode 501‐residue M, 55882 and 388‐residue M, 43431 proteins, respectively. These genes were expressed in vitro and their initiation codons verified by deletion analysis. Tn5–751 had inserted near the centre of the exeE gene in the L1.97 strain. Subclones of the Kpnl 4.1 kb fragment which contained only the exeE gene fully complemented the mutation, indicating that its function is required both for extracellular secretion and outer membrane assembly. ExeE and ExeF are highly similar to other proteins which have been shown to be involved in extracellular secretion, suggesting that an additional export apparatus beyond that required for inner membrane translocation may be part of the physiology of many Gram‐negative bacteria.
Expression of extracellular phospholipase from Serratia liquefaciens is growth‐phase‐dependent, catabolite‐repressed and regulated by anaerobiosis
Authors: Givskov Michael , Molin Søren
Published: October 2006

Many members of the genus Serratia synthesize and excrete a number of extracellular hydrolytic enzymes. One of these is the phospholipase A1 from Serratia liquefaciens, the expression of which is growth‐phase‐dependent. Through the use of gene fusions and primer extension analysis we show that the expression of phospholipase is subject to positive transcriptional regulation of a dual promoter system; one promoter positioned approximately 600bp upstream from the phIA gene is responsible for the induction of phospholipase expression under anaerobic conditions, and the other promoter positioned 50bp upstream from the phIA gene is subject to catabolite repression and induced during the transition from exponential to late log‐phase of bacterial growth. On the basis of sequence homology and behaviour in the relevant Escherichia coli mutants, we suggest the distant promoter to be Fnr‐controlled and the proximal phIA promoter to be a member of the FIbB‐controlled flagellar‐chemotaxis regulon.
Mip protein of Legionella pneumophila exhibits peptidyl‐prolyl‐cis/trans isomerase (PPIase) activity
Authors: Fischer Gunter , Bang Holger , Ludwig Birgit , Mann Karlheinz , Hacker Jörg
Published: October 2006

Legionella pneumophila is an intracellular parasite which is able to survive and multiply in human monocytes and alveolar macrophages. The Mip (macro‐phage infectivity potentiator) protein has been shown to be an essential virulence factor. A search of translated nucleic acid data bases has shown that the Mip protein from strain Wadsworth possesses regions homologous to those found in the FK506‐binding proteins (FKBPs) of several different eukaryotic organisms. FKBPs are able to bind to the immunosuppressant macrolide FK506 and possess peptidyl‐prolyl cis/trans isomerase (PPIase) activity. The gene coding for the Mip protein was cloned from the chromosome of L. pneumophila strain Philadelphia I and sequenced. It was synthesized in Escherichia coli K‐12 and after purification it exhibited PPIase activity catalysing the slow cis/trans isomerization of prolyl peptide bonds in oligopeptides. Mip is inhibited by FK506 and fully resistant to cyclosporin A, as was also found for the recently characterized FKBP‐type PPlases of eukaryotes. However, the N‐terminal extension of Mip and/or the substitutions of the variable amino acids in the C‐terminal FKBP core lead to variations, when compared with eukaryotic FKBPs, in substrate specificity with the oligopeptide substrates of type Suc‐Ala‐Xaa‐Pro‐Phe‐4‐nitroanilide. Nevertheless, the Legionella Mip factor represents a bacterial gene product which shares some characteristics normally found in eukaryotic proteins. In view of the activity of PPIases in protein‐folding reactions, such prokaryotic FKBP analogues may represent a new class of bacterial pathogenicity factors.
MicroReview Control of translation initiation in Saccharomyces cerevisiae
Authors: Yoon Heejeong , Donahue Thomas F.
Published: October 2006

The first observations regarding the control of translation initiation in the yeast Saccharomyces cerevisiae were made by Fred Sherman and his colleagues in 1971. Elegant genetic studies of the CYC1 gene resulted in the formulation of ‘Sherman's Rules’ for translation initiation as follows: (i) AUG is the only initiator codon. (ii) the most proximal AUG from the 5’end of a message will serve as the start site of translation; and (iii) if the upstream AUG codon is mutated then initiation begins at the next available AUG in the message. Hidden within these rules is the mechanism of eukaryotic translation initiation, as these very same rules were later shown to apply to higher eukaryotic organisms and were formulated into the scanning model. However, only in the past five years has yeast been taken seriously as an organism for studying the mechanism of eukaryotic translation initiation. The basis for this is that the yeast genes for at least four mammalian translation initiation factor homologues have been identified and the number is growing. Similar factors suggest similar mechanisms for translation initiation between yeast and mammals. For some translation initiation factors, the genetics of yeast has provided new insights into their function. A mechanism for regulating translation initiation in mammalian cells is now evident in yeast. It seems clear that the molecular genetics of yeast coupled with the available in vitro translation system will provide a wealth of information in the future regarding translational control and regulatory mechanisms. The purpose of this review is to summarize what is known about translational control in S. cerevisiae.
Genomic diversity and organization of virulence genes in the pathogenic anaerobe Clostridium perfringens
Authors: Canard B. , Saint‐Joanis B. , Cole S. T.
Published: October 2006

Pulsed‐field gel electrophoresis has been used to assess genomic diversity and to identify virulence regions in 10 strains, representing all five serotypes, of the anaerobic pathogen Clostridium perfringens. Detailed physical and gene maps of the 3.6 Mb circular chromosomes have been established in eight cases and used to deduce a consensus map. With one exception the chromosomal arrangement was relatively constant and map comparison allowed three hypervariable regions to be identified. One of these was associated with the enterotoxin gene, cpe, which is an important cause of human diarrhoea following the ingestion of food contaminated with C. perfringens. Another variable region spanning the major virulence gene plc, which encodes the cytolytic toxin, α, was located near oriC in all cases whereas the gene for another lethal typing toxin, ɛ, was borne by an episome. It now seems likely that the serological variations, and the changes in the pathogenic spectrum which constitute the C. perfringens typing system, may be due entirely to the loss, or acquisition, of extrachromosomal genetic elements.
Transcriptional regulation of the plastocyanin and cytochrome C553 genes from the cyanobacterium Anabaena species PCC 7937
Authors: Bovy Arnaud , de Vrieze Geert , Borrias Miles , Weisbeek Peter
Published: October 2006

The effect of copper on the levels of plastocyanin (PC) and cytochrome c553 (cyt‐c)‐specific transcripts from Anabaena sp. PCC 7937 was investigated. The addition of copper resulted in a marked increase in PC mRNA levels, and a decrease in cyt c mRNA levels. Thus the functional exchange between PC and cyt c seems to be regulated at the mRNA level. The copper‐dependent increase in PC and decrease in cyt c mRNA levels was abolished when chloramphenicol was added to the cells. This suggests that de novo synthesis of at least one trans‐acting element is required to regulate PC and cyt c mRNA levels. Both PC and cyt c mRNA stability was found to be unaltered under varying Cu2+ regimes. This leads to the conclusion that expression of both genes is regulated at the level of initiation of transcription.
Cloning of genes affecting capsule expression in Staphylococcus aureus strain M
Authors: Lee Chia Y.
Published: October 2006

Staphylococcus aureus strain M produces large amounts of capsular polysaccharide. It produces a non‐encapsulated variant at a frequency of 0.01% at 37° C. At high temperature (43° C), the frequency of capsule loss was shown to be 1–38%. A 19kb plasmid and a prophage were found to be carried by the M strain, but curing of these elements did not affect capsular production. To clone the capsular (cap) genes, a plasmid library of S. aureus M was constructed directly in S. aureus RN4200. The library was then infected with phage 80α. After transduction of the phage lysates to a Cap‐ mutant derived from M strain, a recombinant plasmid was obtained which complemented the mutant to a Cap+ phenotype. Chromosomal walking experiments were used to clone additional nearby cap genes. Complementation tests using a collection of Cap‐ mutants showed that most of the mutants were complemented by a 19.4 kb DNA fragment, suggesting that the majority of the cap genes affecting capsule production are clustered together.
Co‐ordinate expression of virulence genes during swarm‐cell differentiation and population migration of Proteus mirabilis
Authors: Ariison Clive , Lai Hsin‐Chih , Hughes Colin
Published: October 2006

The uropathogenic Gram‐negative bacterium Proteus mirabilis exhibits a form of multicellular behaviour termed swarming, which involves cyclical differentiation of typical vegetative cells into filamentous, multi‐nucleate, hyperflagellate swarm cells capable of rapid and co‐ordinated population migration across surfaces. We observed that differentiation into swarm cells was accompanied by substantial increases in the activities of intracellular urease and extracellular haemolysjn and metalloprotease, which are believed to be central to the pathogenicity of P. mirabills. In addition, the ability of P. mirabilis to invade human urothelial cells in vitro was primarily a characteristic of differentiated swarm cells, not vegetative cells. These virulence factor activities fell back as the cells underwent cyclical reversion to the vegetative form (consolidation), in parallel with the diagnostic modulation of flagellin levels on the cell surface. Control cellular alkaline phosphatase activities did not increase during differentiation or consolidation. Non‐flagellated, non‐motile transposon insertion mutants were unable to invade urothelial cells and they generated only low‐level activities of haemolysin, urease and protease (0–10% of wild type). Motile mutants unable to differentiate into swarm cells were comparably reduced in their haemolytic, ureolytic and invasive phenotypes and generated threefold less protease activity. Mutants that were able to form swarm cells but exhibited various aberrant patterns of swarming migration produced wild‐type activities of haemotysin, urease and protease, but their ability to enter urothelial cells was three‐ to 10‐fold lower. Analysis of haemolysin (hpmA) transcripts showed that during swarm cell differentiation there was a c. 50‐fold increase in the level of the predicted major 5.2 kb and minor 6.9 kb mRNAs transcribed from the hpm operon, and assay of mRNA complimentary to urease (ureC) and flagellin (fliC) gene sequences confirmed that modulation of virulence factor activity during the swarming cycle resulted from differential expression of virulence genes in parallel with fliC gene expression. Hybridization of stage‐specific mRNA with 30 random, non‐overlapping chromosomal gene probes provided no evidence for universal changes in the expression of the P. mirabiis genome, suggesting that differential virulence gene expression may be a specific strategy.
gyrB mutations which confer coumarin resistance also affect DNA supercoiling and ATP hydrolysis by Escherichia coli DNA gyrase
Authors: Confreres Asunción , Maxwell Anthony
Published: October 2006

Coumarins are inhibitors of the ATP hydrolsis and DNA supercoiling reactions catalysed by DNA gyrase. Their target is the B subunit of gyrase (GyrB), encoded by the gyrB gene. The exact mode and site of action of the drugs is unknown. We have identified four mutations conferring coumarin resistance to Escherichia coli: Arg‐136 to Cys, His or Ser and Gly‐164 to Val. In vitro, the ATPase and supercoiling activities of the mutant GyrB proteins are reduced relative to the wild‐type enzyme and show resistance to the coumarin antibiotics. Significant differences in the susceptibility of mutant GyrB proteins to inhibition by either chlorobiocin and novobiocin or coumermycin have been found, suggesting wider contacts between coumermycin and GyrB. We discuss the significance of Arg‐136 and Gly‐164 in relation to the notion that coumarin drugs act as competitive inhibitors of the ATPase reaction.
A Salmonella dublin virulence plasmid locus that affects bacterial growth under nutrient‐limited conditions
Authors: Pullinger Gillian D. , Lax Alistair J.
Published: October 2006

This paper reports the characterization of a new locus, vagC/vagD, on the virulence plasmid of Salmonella dublin. Strain G19, harbouring a TnA insertion in vagC, exhibited reduced virulence although vagC was outside the 8 kb essential virulence region. G19 was also unable to grow on minimal‐medium containing various sole carbon/energy sources, unlike the wild‐type and plasmid‐cured strains. Sequencing of the locus revealed the presence of two ORFs (vagC and vagD) which overlapped by one nucleotide. The VagC polypeptide (12 kDa) was observed using minicell expression. Results indicated that vagD was responsible for the pheno‐typic differences observed between the wild type and G19, and that vagC modulated the activity of vagD. Furthermore, microscopic analysis of G19 cells harvested from minimal‐medium plates showed that a high proportion of cells were elongated, which suggested that vagC and vagD might be involved in coordination of plasmid replication with cell division. We propose that vagD, under certain environmental conditions, acts to prevent cell division until plasmid replication is complete, thus aiding plasmid maintenance. vagC and vagD are absent from the related virulence plasmid of Salmonella typhimurium.
petR, located upstream of the fbcFBC operon encoding the cytochrome bc1 complex, is homologous to bacterial response regulators and necessary for photosynthetic and respiratory growth of Rhodobacter capsulatus
Authors: Tokito Mariko K. , Daldal Fevzi
Published: October 2006

Interposon mutagenesis of a region upstream of the petABC(fbcFBC) operon, encoding the ubiquinol: cytochrome c2 oxidoreductase (bc1 complex) of the photosynthetic bacterium Rhodobacter capsulatus revealed the presence of two genes, petP and petR. DNA nucleotide sequence determination of this region indicated that petP and petR are transcribed In the same direction as the petABC(fbcFBC) operon, and are translationally coupled. A silent insertion located In the interoperonal region separating petPR and the petABC(fbcFBC) genes indicated that these clusters have separate promoters. The deduced amino acid sequence of the putative petR gene product is homologous to various bacterial response regulators, especially to those of the OmpR subgroup. Moreover, it was found that PetR‐ mutants are unable to grow on rich or minimal media by either photosynthesis or respiration, demonstrating that these gene products are essential for growth of R. capsulatus.
Escherichia coli integration host factor stabilizes bacteriophage Mu repressor interactions with operator DNA in vitro
Authors: Alazard R. , Bétermier M. , Chandler M.
Published: October 2006

Using gel retardation and DNase I protection techniques, we have demonstrated that the Escherichia coli integration host factor (IHF) stabilizes the interaction between Mu repressor and its cognate operator‐binding sites in vitro. These results are discussed in terms of a model in which IHF may commit the phage to the lytic or lysogenic pathway depending on the occupancy of the operator sites by the repressor.
Stabilization of bacteriophage Mu repressor‐operator complexes by the Escherichia coli integration host factor protein
Authors: Gama Maria‐José , Toussaint Ariane , Higgins N. Patrick
Published: October 2006

All of the previously described effects of integration host factor (IHF) on bacteriophage Mu development have supported the view that IHF favours transposition‐replication over the alternative state of lysogenic phage growth. In this report we show that, consistent with a model in which Mu repressor binding to its operators requires a particular topology of the operator DNA, IHF stimulates repressor binding to the Ol and O2 operators and enhances Mu repression. IHF would thus be one of the keys, besides supercoiling and the H‐NS protein, that lock the operator region into the appropriate topological conformation for high‐affinity binding not only of the phage transposase but also of the phage repressor.
Isolation and molecular characterization of a novel broad‐host‐range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from Gram‐positive organisms
Authors: Antoine Rudy , Locht Camille
Published: October 2006

A 2.6kb plasmid, named pBBR1, was isolated from Bordetella bronchiseptica S87. After insertion of an antibiotic resistance marker, this plasmid could be transferred into Escherichia coli, Bordetella pertussis, B. bronchiseptica, Vibrio cholerae, Rhizobium meliloti, and Pseudomonas putida by transformation or conjugation. Conjugation was possible only when the IncP group transfer functions were provided in trans. As shown by incompatibility testing, pBBR1 does not belong to the broad‐host‐range IncP, IncQ or IncW groups. DNA sequence analysis revealed two open reading frames: one was called Rep, involved in replication of the plasmid, and the other, called Mob, was involved in mobilization. Both the amino‐termtnal region of Mob and its promoter region show sequence similarities to Mob/Pre proteins from plasmids of Gram‐positive bacteria. In spite of these sequence similarities, pBBR1 does not replicate via the rolling‐circle mechanism commonly used by small Gram‐positive plasmids. We therefore speculate that pBBR1 may combine a mobilization mechanism of Gram‐positive organisms with a replication mechanism of Gram‐negative organisms. Determination of the plasmid copy number in E. coli and B. pertussis indicated that pBBR1 has a rather high copy number, which, in conjunction with its small size and broad host range, renders it paricularly interesting for studies of broad‐host‐range replicons and for the development of new cloning vectors for a wide range of Gram‐negative bacteria.
Phylogenetic relationships among the agent of bacillary angiomatosis, Bartonella bacilliformis, and other alpha‐proteobacteria
Authors: Relman David A. , Lepp Paul W. , Sadler Kay N. , Schmidt Thomas M.
Published: October 2006

Bacillary angiomatosis (BA) and chronic bartonellosis are bacterial infections of humans which result in an unusual vascular proliferative tissue response. In order to determine their phylogenetic relationships, we have determined greater than 95% of the 16S rRNA sequences for these two organisms by amplification directly from infected BA tissue and from a Bartonella bacilliformis lyophilized culture. The BA agent and B. bacilliformis are closely related alpha‐proteobacteria (98.5%), although the BA agent is more closely related to Rochalimaea quintana (99.1%). Contrary to previous belief, the BA agent is distinct from, and less closely related to, the cat scratch bacillus (Afipia fells) (90.7%). We propose a novel secondary structure in a hypervariable region of the 16S rRNA which is useful for alignment of primary sequences and which may be useful for design of nucleic acid probes.
Expression of the Escherichia coli dam gene
Authors: Løbner‐Olesen Anders , Boye Erik , Marinus M. G.
Published: October 2006

The Escherichia coli dam gene and upstream sequences were cloned from the Kohara phage 4D4. Five promoters were found to contribute to dam gene transcription. PI and P2 (the major promoter) were situated approximately 3.5 kb upstream of the structural gene, P3 was within the aroB gene, P4 was within the urf74.3 gene, and P5 was in the urf74.3‐dam intergenic region. The nucleotide sequence of 2280 bp of DNA containing P1 and P2, was determined and shown to have the potential to encode a protein of approximately 16 kDa between P1, P2, and the aroB gene. This 16 kDa open reading frame has been Identified as aroK, the gene for shikimic acid kinase I. Thus the dam gene is part of an operon containing aroK, aroB, urf74.3, and dam. The transcriptional start points of the promoters were determined. A comparison of their nucleotide sequences suggested that P1‐P4 were all recognized by the σ70 subunit of the RNA polymerase.
DNA twist as a transcriptional sensor for environmental changes
Authors: Wang Jian‐Ying , Syvanen Michael
Published: October 2006

A variety of reports describe shifts in the environment which cause a corresponding change in the measured linking number of plasmid DNA isolated from bacterial cells. This change in linking number is often attributed to a change in superhelical density. This, coupled with the observation that transcription is often dependent upon the superhelical density of the DNA template seen in vitro, has led to the suggestion that superhelical density may control expression of certain genes. However, since many environmental changes could, in principle, influence DNA twist itself, then the measured differences in linking number, δLk, may simply be a consequence of variation in twist according to the relationship δLk=δTw+δWr, where δTw and δWr are changes in twist and writhe, respectively. In fact, we show that when an environmental change causes a change in the helical pitch of the DNA, and if the superhelical density of DNA is regulated to remain constant according to the homeostatic model of Menzel and Gellert, then δLkδTw. We have found that there are a number of published reports describing variation in promoter activity as a function of linking number that can be explained by considering twist. We suggest that there are classes of σ70 promoters whose ability to be recognized by RNA polymerase is exquisitely sensitive to the relative orientation of the ‐35 and ‐10 regions, and environmental conditions can control this relative orientation by changing DNA twist. The recA and proU promoters which are activated by cold shock and osmotic shock, respectively, behave as if they are twist‐sensitive promoters. Consideration of DNA twist can also account for the change in activity of a number of other promoters when they are placed in bacterial strains defective in either DNA gyrase or topoisomerase.
A developmentally regulated cysteine proteinase gene of Leishmania mexicana
Authors: Mottram Jeremy C. , Robertson Colin D. , Coombs Graham H. , Barry J. David
Published: October 2006

We have isolated a gene encoding a previously unreported class of trypanosomatid cysteine proteinase (CP) from the protozoan parasite Leishmania mexicana. The single‐copy gene (Imcpa) has several unusual features that distinguish it from CP genes cloned from the related species Trypanosoma brucei and Trypanosoma cruzi. These include a shorter C‐terminal extension of only 10 amino acids and a three‐amino‐acid insertion, GlyValMet, close to the predicted N‐terminus of the mature protein. Northern blot analysis showed that the gene is expressed in all life‐cycle stages but at higher levels in the amastigote stage in the mammal and in stationary phase promastigote cultures which contain the infective meta‐cyclic form of the parasite. A precursor protein of 38 kDa was detected in amastigotes and stationary phase promastigotes with antisera specific to the LmCPa pro‐region, but was barely detectable in early log‐phase promastigotes. Anti‐central domain antisera recognized the 38 kDa precursor and 24 and 27 kDa proteins. The major CPs of L. mexicana amastigotes, previously designated types A, B and C, were not detected with the antisera, suggesting that the gene codes for a previously uncharacterized CP in L. mexicana. The 24 kDa protein detected by the antiserum has no activity towards gelatin but apparently hydrolyses the peptide substrate BzPhe‐ValArgAMC. The relative levels of the 24 and 27 kDa proteins vary between the different life‐cycle stages. The results indicate that expression of this CP is regulated at both the RNA and protein level.
Evasion of type I and type II DNA restriction systems by Incl1 plasmid Collb‐P9 during transfer by bacterial conjugation
Authors: Read Timothy D. , Thomas Angela T. , Wilkins Brian M.
Published: October 2006

Transmission of unmodified plasmid Collb‐P9 by bacterial conjugation is markedly resistant to restriction compared with transfer by transformation. One process allowing evasion of type I and II restriction systems involves conjugative transfer of multiple copies of the plasmid. A more specialized evasion mechanism requires the Ard (alleviation of restriction of DNA) system encoded by Collb. The ard gene is transferred early in conjugation and specifically alleviates DNA restriction by all known families of type I enzyme, including Eco K. Collb has no effect on EcoK modification but this activity is impaired by multicopy recombinant plasmids supporting overexpression of ard. Genetic evidence shows that Ard protects Collb from Eco K restriction following conjugative transfer and that this protection requires expression of the gene on the immigrant plasmid. It is proposed that carriage of ard facilitates transfer of Collb between its natural enterobacterial hosts and that the route of DNA entry is important to the restriction‐evasion mechanism.
Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin
Authors: Gilbert H. J. , Hazlewood G. P. , Laurie J. I. , Orpin C. G. , Xue G. P.
Published: October 2006

A cDNA (xynA), encoding xylanase A (XYLA), was isolated from a cDNA library, derived from mRNA extracted from the rumen anaerobic fungus, Neocallimastix patriciarum. Recombinant XYLA, purified from Escherichia coli harbouring xynA, had a Mr, of 53000 and hydrolysed oat‐spelt xylan to xylobiose and xylose. The enzyme did not hydrolyse any cellulosic substrates. The nucleotide sequence of xynA revealed a single open reading frame of 1821 bp coding for a protein of Mr, 66192. The predicted primary structure of XYLA comprised an N‐terminal signal peptide followed by a 225‐amino‐acid repeated sequence, which was separated from a tandem 40‐residue C‐terminal repeat by a threonine/proline linker sequence. The large N‐terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full‐length enzyme. The reiterated structure of XYLA suggests that the enzyme was derived from an ancestral gene which underwent two discrete duplications. Sequence comparison analysis revealed significant homology between XYLA and bacterial xylanases belonging to cellulase/xylanase family G. One of these homologous enzymes is derived from the rumen bacterium Ruminococcus flavefaciens. The homology observed between XYLA and a rumen prokaryote xylanase could be a consequence of the horizontal transfer of genes between rumen prokaryotes and lower eukaryotes, either when the organisms were resident in the rumen, or prior to their colonization of the ruminant. It should also be noted that Neocallimastix XYLA is the first example of a xylanase which consists of reiterated sequences. It remains to be established whether this is a common phenomenon in other rumen fungal plant cell wall hydrolases.
Cell division in Escherichia coli minB mutants
Authors: Akerlund Thomas , Bernander Rolf , Nordström Kurt
Published: October 2006

In Escherichia coli minB mutants, cell division can take place at the cell poles as well as non‐polarly in the cell. We have examined growth, division patterns, and nucleoid distribution in individual cells of a minC point mutant and a minB deletion mutant, and compared them to the corresponding wild‐type strain and an intR1 strain in which the chromosome is overreplicated. The main findings were as follows. In the minB mutants, polar and non‐polar divisions appeared to occur independently of each other. Furthermore, the timing of cell division in the cell cycle was found to be severely affected. In addition, nucleoid conformation and distribution were considerably disturbed. The results obtained call for a re‐evaluation of the role of the MinB system in the E coli cell cycle, and of the concept that limiting quanta of cell division factors are regularly produced during the cell cycle.
The stable BRP signal peptide causes lethality but is unable to provoke the translocation of cloacin DF13 across the cytoplasmic membrane of Escherichia coli
Authors: van der Wal F. J. , Oudega B. , Kater M. M. , ten Hagen‐Jongman C. M. , de Graaf F. K. , Luirink J.
Published: October 2006

The bacteriocin release protein (BRP) mediates the secretion of cloacin DF13. The BRP precursor is slowly processed to yield the mature BRP and its stable signal peptide which is also involved in cloacin DF13 secretion. The function of the stable BRP signal peptide was analysed by constructing two plasmids. First, the stable BRP signal peptide was fused to the murein lipoprotein and, second, a stop codon was introduced after the BRP signal sequence. Exchange of the unstable murein lipoprotein signal peptide for the stable BRP signal peptide resulted in an accumulation of precursors of the hybrid murein lipoprotein. This indicated that the BRP signal peptide, as part of this hybrid precursor, is responsible for the slow processing. The stable BRP signal peptide itself was not able to direct the transfer of cloacin DF13 into the periplasmic space or into the culture medium. Over‐expression of the BRP signal peptide was lethal and caused ‘lysis’. Subcellular fractionation experiments revealed that the BRP signal peptide is located exclusively in the cytoplasmic membrane whereas the mature BRP, targeted by either the stable BRP signal peptide or the unstable Lpp signal peptide, is located in both the cytoplasmic and outer membrane.

These results are in agreement with the hypothesis that the stable signal peptide and the mature BRP together are required for the passage of cloacin DF13 across the cell envelope.
Expression and mutational analysis of the nucleoid‐associated protein H‐NS of Salmonella typhimurium
Authors: Hinton Jay C. D. , Santos Diogenes S. , Seirafi Alexander , Hulton Christopher S. J. , Pavitt Graham D. , Higgins Christopher F.
Published: October 2006

The H‐NS (H1) protein is a major component of bacterial chromatin. Mutations in the hns (osmZ) gene encoding H‐NS are highly pleiotropic, affecting the expression of many unrelated genes in an allele‐specific manner. H‐NS expression was found not to vary with growth phase or growth medium osmolarity. Additionally, 10 independent hns mutations were isolated and characterized. Five of these mutations were the result of an IS10 insertion, each generating a truncated polypeptide. The other five mutations were the same specific deletion of one amino acid, δla46. The various hns mutations exhibited different phenotypes and influenced ONA topology to variable extents. Implications for the mechanism by which H‐NS influences gene expression are discussed.
The plasmid‐encoded chloramphenicol‐resistance protein of Rhodococcus fascians is homologous to the transmembrane tetracycline efflux proteins
Authors: Desomer J. , Vereecke D. , Crespi M. , Van Montagu M.
Published: October 2006

The nucleotide sequence of the chloramphenicol‐resistance gene (cmr) of Rhodococcus fascians NCPPB 1675 (located on the conjugative plasmid pRF2) allowed the identification of two possible open reading frames (ORFs), of which ORF1 was consistent with the mutational analysis. Biochemical analysis of cmr revealed that it does not encode an antibiotic‐modifying enzyme. The amino acid sequence of 0RF1 predicted a hydrophobic protein, with 12 putative membrane‐spanning domains, homologous to proteins involved in the efflux of tetracycline across the plasma membrane. Expression of the cmr gene was induced by addition of chloramphenicol to the growth media. The promoter of this gene was restricted to 50 bp upstream from a 200 bp 5′‐untrans‐lated mRNA region, the latter containing two inverted repeats. At the amino acid level, the cmr gene is 52% identical to a previously identified chloramphenicol‐resistance determinant in Streptomyces lividans, indicating a wider dispersion of this type of cmr gene among the actinomycetes.
Identification and characterization of virK, a virulence‐associated large plasmid gene essential for intercellular spreading of Shigella flexneri
Authors: Nakata N. , Sasakawa C. , Okada N. , Tobe T. , Fukuda I. , Suzuki T. , Komatsu K. , Yoshikawa M.
Published: October 2006

Seven virulence loci have been identified by Tn5 insertion mutagenesis on the large 230 kb plasmid (pMYSH6000) of Shigella flexneri 2a. In this study, we used Tn10 insertion mutagenesis and identified a novel virulence locus on pMYSH6000 responsible for bacterial spread. Characterization of the invading bacteria of the Tn10 insertion mutants in the epithelial cells revealed that the bacteria were capable of at least some intracellular spreading but not intercellular spreading. Immunoblot analysis of lysates of the Tn10 insertion mutants with a VirG‐specific antipeptide antibody revealed diminished levels of the 116 kDa VirG protein. The virG mRNA in the mutants, however, was expressed at the same level as that in the wild type. The DNA region required for the virulence phenotype was localized to a 1.6 kb DNA sequence in the Sal I‐K fragment on the plasmid, and thus the locus was designated virK. Expression of virK in Escherichia coli using a T7 RNA polymerase‐dependent promoter system yielded a 36 kDa protein. The nucleotide sequence of 1642 bp encoding VirK function was determined, and an open reading frame encoding 316 amino acid residues was shown to encode the VirK protein. The virK region was highly conserved among the large virulence plasmids of shigellae and enteroinvasive Escherichia coli. These results suggest that VirK function is an essential virulence determinant for shigellae Involved in the expression of virG gene product at post‐transcriptional level.
Analysis of the transcriptional activity of the hut promoter in Bacillus subtilis and identification of a cis‐acting regulatory region associated with catabolite repression downstream from the site of transcription
Authors: Oda M. , Katagai T. , Tomura D. , Shoun H. , Hoshino T. , Furukawa K.
Published: October 2006

Levels of transcripts initiated at a hut promoter in Bacillus subtilis were analysed. The addition of histidine to the culture medium increased the level of the transcript sixfold. In the presence of histidine and glucose together, the level of the transcript was reduced to the level in the absence of Induction. Furthermore, addition of a mixture of 16 amino acids to cultures of induced cells and of catabolite‐repressed cells decreased levels of the transcript 16‐fold and 2.6‐fold, respectively. Thus, it appears that at least three regulatory mechanisms associated with induction, catabolite repression, and amino acid repression, control the transcriptional activity of the hut promoter. Expression of the hut promoter–lacZ fusions that contained various regions of the hutP gene and deletion analysis of the hutP region revealed a cis‐acting sequence associated with catabolite repression that was located between positions +204 and +231 or around position +203.
Antibodies to N‐terminal peptides of gonococcal porin are bactericidal when gonococcal lipopolysaccharide is not sialylated
Authors: Elkins Christopher , Carbonetti Nicholas H. , Varela Victor A. , Stirewalt Derek , Klapper David G. , Sparling P. Frederick
Published: October 2006

Six synthetic 25‐mer peptides corresponding to certain presumed surfaced‐exposed regions of gonococcal porin protein I (PI) were made from strains FA19 (PIA) and MS11 (PIB). Four peptides were immunogenic in rabbits. Affinity‐purified antisera against both PIA and PIB N‐terminal peptides were bactericidal for homologous gonococci and many heterologous PI serovars. However, sialylation of gonococcal lipopolysaccharide (LPS) by growth of gonococci in the presence of cytidine monophosphate–neuraminic acid (CMP–NANA) abrogated the bactericidal activity of these antisera. Binding of anti‐PI monoclonal antibodies to whole gonococci was reduced two‐ to fourfold by sialylation of LPS, suggesting that sialylation may inhibit bactericidal activity by masking porin epitopes. However, binding of anti‐PII (Opa) monoclonal antibodies was not inhibited, yet complement‐mediated killing was inhibited by sialylated LPS. Binding of complement components C3 and C9 was inhibited in the presence of either anti‐PI or anti‐PII monoclonals when gonococci were grown in the presence of CMP–NANA. Thus sialylation inhibited both anti‐PI antibody binding and complement deposition, with a resultant decrease in bactericidal activity.
Control of gene expression in the temperate coliphage 186. X. The cl repressor directly represses transcription of the late control gene B
Authors: Dibbens Justin A. , Gregory Stephen L. , Egan J. Barry
Published: October 2006

We have found that the repressor of 186 lytic transcription, CI, represses transcription of the late control gene B, with no involvement of the B protein Itself. In clone studies we showed that CI repressed transcription from the B promoter and that temperature inactivation of CIts led to B derepression. We conclude that CI repressor directly represses transcription of the Bgene and, with prophage induction, it is probable that the inactivation of the CI repressor not only derepresses early lytic transcription, but also derepresses B gene transcription, leading to the activation of transcription from the late promoters.