bluuebunny/crossref_metadata_2025 huggingface dataset:
A distinct 5′ flanking var gene region regulates Plasmodium falciparum variant erythrocyte surface antigen expression in placental malaria
Authors: Vázquez‐Macías Aleida , Martínez‐Cruz Perla , Castañeda‐Patlán María Cristina , Scheidig Christine , Gysin Jürg , Scherf Artur , Hernández‐Rivas Rosaura
Published: March 2003

The Plasmodium falciparum multigene var family codes for approximately 50 variant adhesive proteins expressed in a mutually exclusive manner at the surface of infected red blood cells (iRBCs). Switching expression of var genes can lead to fundamental changes in the adhesive and antigenic properties of iRBCs. For example, a specific phenotypic switch in adhesion from CD36 to chondroitin sulphate A (CSA) is associated with malaria pathogenesis in pregnant women. The factors and DNA elements that control the expression of a particular member of the var gene family during gestational malaria remains enigmatic. Here, we report that the subtelomeric FCR3 varCSA is expressed under the control of a unique DNA element of 1.8 kb, whereas the other members of the var multigene family are flanked by common regulatory elements. The 5′varCSA‐type element is conserved as a single copy in laboratory strains and clinical isolates from Brazil and West Africa and contains two distinct repetitive elements of 150 bp and 60 bp respectively. The 5′varCSA‐type sequence tags a var gene in the 3D7 genome that is homologous to the FCR3 varCSA gene. A recombinant DBLγ domain of this var gene showed specific binding to CSA. This subtelomeric varCSA gene is transcribed in the opposite sense when compared with the usual orientation of telomere‐adjacent var genes. This unique arrangement might explain why the varCSA gene is relatively conserved in genetically distinct parasites despite being located in a highly recombinogenic chromosome compartment. The 5′ untranslated region (UTR) of the varCSA‐type sequence is also transcribed in placental isolates that bind to CSA, illustrating an important role for the unique 5′varCSA‐type sequence in the regulation of var genes involved in malaria pathogenesis in pregnant women. However, this promoter is not always found to be transcribing var genes selected for expression of products that bind to CSA in vitro. Our work identifies a sequence tag for the identification of varCSA genes in placental isolates for the first time.
Activation of the Escherichia coli nfnB gene by MarA through a highly divergent marbox in a class II promoter
Authors: Barbosa Teresa M. , Levy Stuart B.
Published: March 2003

MarA is a global regulator that mediates resistance to multiple environmental hazards such as antibiotics, disinfectants and oxidative stress agents by modulating the expression of a large number of genes in the Escherichia coli chromosome. Two E. coli MarA homologues, SoxS and Rob also control, to different extents, genes in the mar/sox/rob regulon. The controlling element for these proteins is a 20 bp ‘marbox’ sequence in the promoter region of regulated genes. Using in vitro assays and mutagenesis of promoter fusions in whole cells, we identified the cis regulatory element involved in MarA upregulation of the oxygen‐insensitive nitroreductase nfnB gene. MarA binds to a marbox that is highly divergent from the previously proposed consensus (eight differences out of 14 specified nucleotides). Although purified SoxS and Rob proteins, like MarA, activated nfnB transcription in vitro, only constitutive expression of chromosomal marA, but not of soxS and rob genes, affected nfnB expression in whole cells. Increased expression, but limited as compared with MarA, was only achieved by plasmid‐mediated overexpression of SoxS and Rob. This study shows that MarA can regulate gene expression through a functional marbox that is considerably divergent from the current consensus sequence. The data suggest that MarA is preferred over SoxS and Rob in upregulating nfnB. The findings imply that other different but physiologically important marbox DNA–MarA interactions take place in the regulation of still uncharacterized members of the mar regulon.
Surface antigenic variation in Giardia lamblia
Authors: Nash Theodore E.
Published: March 2003

Giardia lamblia
, a common intestinal dwelling protozoan and a cause of diarrhoea in humans and animals world‐wide, undergoes surface antigenic variation. The variant‐specific surface proteins (VSPs) are a family of related, highly unusual proteins that cover the entire surface of the parasite. VSPs are cysteine‐rich proteins containing many CXXC motifs, one or two GGCY motifs, a conserved hydrophobic tail and a Zn finger motif. The biological role(s) of VSPs is unclear. As VSPs are resistant to the effects of intestinal proteases, they likely allow the organism to survive in the protease‐rich small intestine. Although immune escape is commonly mentioned as the reason antigenic variation occurs, VSP expression changes
in vivo even in the absence of an adaptive immune system suggesting the biological role of antigenic variation is more complex. The molecular mechanisms involved in antigenic variation are not known but appear to differ from those known to occur in other protozoa.
Identification of a gene required for the biosynthesis of ornithine‐derived lipids
Authors: Weissenmayer Barbara , Gao Jun‐Lian , López‐Lara Isabel M. , Geiger Otto
Published: March 2003

Phospholipids are the membrane‐forming constituents in all living organisms. In addition to phosphorus‐containing lipids, the membranes of numerous bacteria contain significant amounts of phosphorus‐free polar lipids, often derived from amino acids. Although lipids derived from the amino acid ornithine are widespread among bacteria, their biosynthesis is unknown. Here, we describe the isolation of mutants of Sinorhizobium meliloti deficient in the biosynthesis of ornithine‐derived lipids (OL). Complementation of such mutants with a sinorhi‐zobial cosmid gene bank, subcloning of the complementing fragment and sequencing of the subclone led to the identification of a gene (olsA) coding for a presumptive acyltransferase. Amplification of this gene and its expression in OL‐deficient mutant backgrounds of S. meliloti demonstrates that it is required for OL biosynthesis. An OL‐deficient mutant of S. meliloti disrupted in olsA shows wild type‐like growth behaviour and is capable of inducing nitrogen‐fixing nodules on legume hosts. A lyso‐ornithine lipid‐dependent acyltransferase activity forming OL requires acyl‐AcpP as the acyl donor and expression of the olsA gene.
Mechanochemical ATPases and transcriptional activation
Authors: Zhang X. , Chaney M. , Wigneshweraraj Siva R. , Schumacher J. , Bordes P. , Cannon W. , Buck M.
Published: March 2003

Transcriptional activator proteins that act upon the σ54‐containing form of the bacterial RNA polymerase belong to the extensive AAA+ superfamily of ATPases, members of which are found in all three kingdoms of life and function in diverse cellular processes, often via chaperone‐like activities. Formation and collapse of the transition state of ATP for hydrolysis appears to engender the interaction of the activator proteins with σ54 and leads to the protein structural transitions needed for RNA polymerase to isomerize and engage with the DNA template strand. The common oligomeric structures of AAA+ proteins and the crea‐tion of the active site for ATP hydrolysis between protomers suggest that the critical changes in protomer structure required for productive interactions with σ54‐holoenzyme occur as a consequence of sensing the state of the γ‐phosphate of ATP. Depending upon the form of nucleotide bound, different functional states of the activator are created that have distinct substrate and chaperone‐like binding activ‐ities. In particular, interprotomer ATP interactions rely upon the use of an arginine finger, a situation reminiscent of GTPase‐activating proteins.
Molecular and functional characterization of kshA and kshB, encoding two components of 3‐ketosteroid 9α‐hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1
Authors: Geize R. van der , Hessels G. I. , Gerwen R. van , Meijden P. van der , Dijkhuizen L.
Published: March 2003

9α‐Hydroxylation of 4‐androstene‐3,17‐dione (AD) and 1,4‐androstadiene‐3,17‐dione (ADD) is catalysed by 3‐ketosteroid 9α‐hydroxylase (KSH), a key enzyme in microbial steroid catabolism. Very limited knowledge is presently available on the KSH enzyme. Here, we report for the first time the identification and molecular characterization of genes encoding KSH activity. The kshA and kshB genes, encoding KSH in Rhodococcus erythropolis strain SQ1, were cloned by functional complementation of mutant strains blocked in AD(D) 9α‐hydroxylation. Analysis of the deduced amino acid sequences of kshA and kshB showed that they contain domains typically conserved in class IA terminal oxygenases and class IA oxygenase reductases respectively. By definition, class IA oxygenases are made up of two components, thus classifying the KSH enzyme system in R. erythropolis strain SQ1 as a two‐component class IA monooxygenase composed of KshA and KshB. Unmarked in frame gene deletion mutants of parent strain R. erythropolis SQ1, designated strains RG2 (kshA mutant) and RG4 (kshB mutant), were unable to grow on steroid substrates AD(D), whereas growth on 9α‐hydroxy‐4‐androstene‐3,17‐dione (9OHAD) was not affected. Incubation of these mutant strains with AD resulted in the accumulation of ADD (30–50% conversion), confirming the involvement of KshA and KshB in AD(D) 9α‐hydroxylation. Strain RG4 was also impaired in sterol degradation, suggesting a dual role for KshB in both sterol and steroid degradation.
Modulation of bft expression by the Bacteroides fragilis pathogenicity island and its flanking region
Authors: Franco Augusto A. , Cheng Rodney K. , Goodman Alan , Sears Cynthia L.
Published: March 2003

To establish a recombinant system for high‐level expression of biologically active Bacteroides fragilis toxin (BFT), we studied the expression of bft in non‐toxigenic B. fragilis (NTBF) strains. The bft gene and the B. fragilis pathogenicity island (BfPAI) were cloned into NTBF strains with two distinct genetic patterns: (i) pattern II, strains lacking the BfPAI and its flanking region; and (ii) pattern III, strains lacking the BfPAI but containing its flanking region. Analysis of BFT activity of these recombinant strains on HT29/C1 cells showed that both the BfPAI and its flanking regions are important to optimal BFT activity. Reverse transcription polymerase chain reaction (RT‐PCR) analysis indicated that the BfPAI and its flanking regions modulate bft expression. Further experiments demonstrated that the ≈ 700 bp region upstream of bft is the BfPAI region critical for optimal bft expression. We conclude that both the region flanking the BfPAI and ≈ 700 bp region upstream of bft are crucial to maximal BFT production by ETBF strains.
Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract
Authors: Bahrani‐Mougeot Farah K. , Buckles Eric L. , Lockatell C. V. , Hebel J. R. , Johnson D. E. , Tang C. M. , Donnenberg M. S.
Published: March 2003

Escherichia coli
is the leading cause of urinary tract infections (UTIs). Despite the association of numerous bacterial factors with uropathogenic
E. coli
(UPEC), few such factors have been proved to be required for UTI in animal models. Previous investigations of urovirulence factors have relied on prior identification of phenotypic characteristics. We used signature‐tagged mutagenesis (STM) in an unbiased effort to identify genes that are essential for UPEC survival within the murine urinary tract. A library of 2049 transposon mutants of the prototypic UPEC strain CFT073 was constructed using mini‐Tn5
km2
carrying 92 unique tags and screened in a murine model of ascending UTI. After initial screening followed by confirmation in co‐infection experiments, 19 survival‐defective mutants were identified. These mutants were recovered in numbers 10
1
‐ to 10
6
‐fold less than the wild type in the bladder, kidneys or urine or at more than one site. The transposon junctions from each attenuated mutant were sequenced and analysed. Mutations were found in: (i) the type 1 fimbrial operon; (ii) genes involved in the biosyn‐thesis of extracellular polysaccharides including group I capsule, group II capsule and enterobacterial common antigen; (iii) genes involved in metabolic pathways; and (iv) genes with unknown function. Five of the genes identified are absent from the genome of the
E. coli
K‐12 strain. Mutations in type 1 fimbrial genes resulted in severely attenuated colonization, even in the case of a mutant with an insertion upstream of the
fim
operon that affected the rate of fimbrial switching from the ‘off’ to the ‘on’ phase. Three mutants had insertions in a new type II capsule biosynthesis locus on a pathogenicity island and were impaired in the production of capsule
in vivo
. An additional mutant with an insertion in
wecE was unable to synthesize enterobacterial common antigen. These results confirm the pre‐eminence of type 1 fimbriae, establish the importance of extracellular polysaccharides in the pathogenesis of UTI and identify new urovirulence determinants.
Coiled‐coil proteins associated with type III secretion systems: a versatile domain revisited
Authors: Delahay Robin M. , Frankel Gad
Published: March 2003

Summary The pathogenic potential of many Gram‐negative bacteria is indicated by the possession of a specialized type III secretion system that is used to deliver virulence effector proteins directly into the cellular environment of the eukaryotic host. Extracellular assemblies of secreted proteins contrive a physical link between the pathogen and host cytosol and enable the translocated effectors to bypass the bacterial and host membranes in a single step. Subsequent interactions of some effector proteins with host cytoskeletal and signalling proteins result in modulation of the cytoskeletal architecture of the aggressed cell and facilitate entry, survival and dissemination of the pathogen. Although the secreted components of type III secretion systems are diverse, many are predicted to share a common coiled‐coil structural feature. Coiled‐coils are ubiquitous and highly versatile assembly motifs found in a wide range of structural and regulatory proteins. The prevalence of these domains in secreted virulence effector proteins suggests a fundamental contribution to multiple aspects of their function, and evidence accumulating from functional studies suggests an intrinsic involvement of coiled‐coils in subunit assembly, translocation and flexible interactions with multiple bacterial and host proteins. The known functional flexibility that coiled‐coil domains confer upon proteins provides insights into some of the pathogenic mechanisms used during interaction with the host.
The dynamic microbe: green fluorescent protein brings bacteria to light
Authors: Southward Carolyn M. , Surette Michael G.
Published: March 2003

The demonstration that the green fluorescent protein (GFP) from the jellyfish Aequorea victoria required no jellyfish‐specific cofactors and could be expressed as a fluorescent protein in heterologous hosts including both prokaryotes and eukaryotes sparked the development of GFP as one of the most common reporters in use today. Over the past several years, the utility of GFP as a reporter has been optimized through the isolation and engineering of variants with increased folding rates, different in vivo stabilities and colour variants with altered excitation and emission spectral properties. One of the great utilities of GFP is as a probe for characterizing spatial and temporal dynamics of gene expression, protein localization and protein–protein interactions in living cells. The innovative application of GFP as a reporter in bacteria has made a significant contribution to microbial cell biology. This review will highlight recent studies that demonstrate the potential of GFP for real‐time analysis of gene expression, protein localization and the dynamics of signalling transduction pathways through protein–protein interactions.
Function in Escherichia coli of the non‐catalytic part of RNase E: role in the degradation of ribosome‐free mRNA
Authors: Leroy Anne , Vanzo Nathalie F. , Sousa Sandra , Dreyfus Marc , Carpousis Agamemnon J.
Published: March 2003

Summary RNase E contains a large non‐catalytic region that binds RNA and the protein components of the Escherichia coli RNA degradosome. The rne gene was replaced with alleles encoding deletions in the non‐catalytic part of RNase E. All the proteins are stable in vivo. RNase E activity was tested using a PT7–lacZ reporter gene, the message of which is particularly sensitive to degradation because translation is uncoupled from transcription. The non‐catalytic region has positive and negative effectors of mRNA degradation. Disrupting RhlB and enolase binding resulted in hypoactivity, whereas disrupting PNPase binding resulted in hyperactivity. Expression of the mutant proteins in vivo anticorrelates with activity showing that autoregulation compensates for defective function. There is no simple correlation between RNA binding and activity in vivo. An allele (rne131), expressing the catalytic domain alone, was put under Plac control. In contrast to rne+, low expression of rne131 severely affects growth. Even with autoregulation, all the mutants are less fit when grown in competition with wild type. Although the catalytic domain of RNase E is sufficient for viability, our work demonstrates that elements in the non‐catalytic part are necessary for normal activity in vivo.
Double‐stranded RNA‐mediated gene silencing of cysteine proteases (falcipain‐1 and ‐2) of Plasmodium falciparum
Authors: Malhotra Pawan , Dasaradhi Palakodeti V. N. , Kumar Amit , Mohmmed Asif , Agrawal Neema , Bhatnagar Raj K. , Chauhan Virander S.
Published: March 2003

Summary Malaria remains a public health problem of enormous magnitude, affecting over 500 million people every year. Lack of success in the past in the development of new drug/vaccines has mainly been attributed to poor understanding of the functions of different parasite proteins. Recently, RNA interference (RNAi) has emerged as a simple and incisive technique to study gene functions in a variety of organisms. In this study, we report the results of RNAi by double‐stranded RNA of cysteine protease genes (falcipain‐1 and ‐2) in the malaria parasite, Plasmodium falciparum. Using RNAi directed towards falcipain genes, we demonstrate that blocking the expression of these genes results in severe morphological abnormalities in parasites, inhibition of parasite growth in vitro and substantial ­accumulation of haemoglobin in the parasite. The inhibitory effects produced by falcipain double‐stranded (ds)RNAs are reminiscent of the effects observed upon administering E‐64, a cysteine protease inhibitor. The parasites treated with falcipain's dsRNAs also show marked reduction in the levels of corresponding endogenous falcipain mRNAs. We also demonstrate that dsRNAs of falcipains are ­broken into short interference RNAs ≈ 25 nucleotides in size, a characteristic of RNAi, which in turn activates sequence‐specific nuclease activity in the malaria parasites. These results thus provide more evidence for the existence of RNAi in P. falciparum and also suggest possibilities for using RNAi as an effective tool to determine the functions of the genes identified from the P. falciparum genome sequencing project.
The DNA site utilized by bacteriophage P22 for initiation of DNA packaging
Authors: Wu Hongyu , Sampson Laura , Parr Ryan , Casjens Sherwood
Published: March 2003

Virion proteins recognize their cognate nucleic acid for encapsidation into virions through recognition of a specific nucleotide sequence contained within that nucleic acid. Viruses like bacteriophage P22, which have partially circularly permuted, double‐stranded virion DNAs, encapsidate DNA through processive series of packaging events in which DNA is recognized for packaging only once at the beginning of the series. Thus a single DNA recognition event programmes the encapsidation of multiple virion chromosomes. The protein product of P22 gene 3, a terminase component, is thought to be responsible for this recognition. The site on the P22 genome that is recognized by the gene 3 protein to initiate packaging series is called the pac site. We report here a strategy for assaying pac site activity in vivo, and the utilization of this system to identify and characterize the site genetically. It is an asymmetric site that spans 22 basepairs and is located near the centre of P22 gene 3.
The LuxR family protein SpnR functions as a negative regulator of N‐acylhomoserine lactone‐dependent quorum sensing in Serratia marcescens
Authors: Horng Yu‐Tze , Deng Su‐Chen , Daykin Mavis , Soo Po‐Chi , Wei Jun‐Rong , Luh Kwen‐Tay , Ho Shen‐Wu , Swift Simon , Lai Hsin‐Chih , Williams Paul
Published: March 2003

Serratia marcescens
SS‐1 produces at least four
N
‐acylhomoserine lactones (AHLs) which were identified using high‐resolution mass spectrometry and chemical synthesis, as
N‐
(3‐oxohexanoyl) homo‐serine lactone (3‐oxo‐C6‐HSL),
N
‐hexanoyl‐ (C6‐HSL),
N
‐heptanoyl (C7‐HSL) and
N
‐octanoyl‐ (C8‐HSL) homoserine lactone. These AHLs are synthesized via the LuxI homologue SpnI, and regulate via the LuxR homologue SpnR, the production of the red pigment, prodigiosin, the nuclease, NucA, and a biosurfactant which facilitates surface translocation.
spnR
overexpression and
spnR
gene deletion show that SpnR, in contrast to most LuxR homologues, acts as a negative regulator.
spnI
overexpression, the provision of exogenous AHLs and
spnI
gene deletion suggest that SpnR is de‐repressed by 3‐oxo‐C6‐HSL. In addition, long chain AHLs antagonize the biosurfactant‐mediated surface translocation of
S. marcescens
SS‐1. Upstream of
spnI
there is a gene which we have termed
spnT
.
spnI
and
spnT
form an operon and although database searches failed to reveal any
spnT homologues, overexpression of this novel gene negatively affected both sliding motility and prodigiosin production.
The gene bolA regulates dacA (PBP5), dacC (PBP6) and ampC (AmpC), promoting normal morphology in Escherichia coli
Authors: Santos Jorge M. , Lobo Mónica , Matos António P. A. , De Pedro Miguel A. , Arraiano Cecilia M.
Published: March 2003

The gene bolA has been shown to trigger the formation of osmotically stable round cells when overexpressed in stationary phase. We show that in poor growth conditions bolA is essential for normal cell morphology in stationary phase and under conditions of starvation. During exponential growth bolA promotes round morphology through a mechanism that is strictly dependent on the two main Escherichia colid,d‐carboxypeptidases, PBP5 and PBP6. The results show that bolA controls the levels of transcription of dacA (PBP5), dacC (PBP6) and ampC (AmpC), a class C β‐lactamase, thus connecting for the first time penicillin binding proteins (PBPs) and β‐lactamases at the level of gene regulation. Furthermore, PBP5 and PBP6 are shown to be independently regulated and to have distinct effects on the peptidoglycan layer. The evidence presented demonstrates that bolA is a regulator of cell wall biosynthetic enzymes with different roles in cell morphology and cell division.
Chaperones of the type III secretion pathway: jacks of all trades
Authors: Page Anne‐Laure , Parsot Claude
Published: March 2003

The type III secretion (TTS) pathway is used by many Gram‐negative bacteria to inject virulence proteins into cells of their host. The activity of the TTS apparatus is controlled by external signals and, in certain conditions, production and secretion are not coupled. Storage of some proteins before secretion involves their association with specific chaperones. Three classes of TTS chaperones have been distinguished according to whether they associate with: (i) one; (ii) several effector proteins; or (iii) the two translocators that allow passage of effectors across the membrane of eukaryotic cells. These chaperones are required for stabilization of their substrate(s) and prevention of their premature interactions with other partners during storage. They also play a role in secretion of their substrate(s). Some chaperones are also involved in transcriptional regulation of certain genes in response to the activity of secretion. The flagellar export apparatus is closely related to the TTS apparatus and some proteins of the flagellar export system have also been proposed to be chaperones that prevent premature interactions between the flagellum subunits.
Differential accumulation of Salmonella[Cu, Zn] superoxide dismutases SodCI and SodCII in intracellular bacteria: correlation with their relative contribution to pathogenicity
Authors: Uzzau Sergio , Bossi Lionello , Figueroa‐Bossi Nara
Published: March 2003

Most Salmonella enterica strains have two peri‐plasmic [Cu, Zn] superoxide dismutases, SodCI and SodCII, encoded by prophage and chromosomal genes respectively. Both enzymes are thought to play a role in Salmonella pathogenicity by intercepting reactive oxygen species produced by the host's innate immune response. To examine the apparent redundancy, we have compared the levels of epitope‐tagged SodCI and SodCII proteins in bacteria growing in vitro, as well as inside tissue culture cells and in mouse tissues. Concomitantly, we have measured the abilities of mutants of either or both sodC genes to proliferate in infected mice in competition assays. Our results show a striking variation in the relative abundance of the two proteins in different environments. In vitro, both proteins accumulate when bacteria enter stationary phase; however, the increase is much sharper and conspicuous for SodCII than for SodCI. In contrast, SodCI vastly predominates in intracellular bacteria where SodCII levels are negligible. In agreement with these findings, most, if not all, of the contribution of [Cu, Zn] superoxide dismutase activity to murine salmonellosis can be ascribed to the SodCI protein. Overall the results of this work suggest that the duplicate sodC genes of Salmonella have evolved to respond to different sets of conditions encountered by bacteria inside the host and in the environment.
The P1 plasmid is segregated to daughter cells by a ‘capture and ejection’ mechanism coordinated with Escherichia coli cell division
Authors: Li Yongfang , Austin Stuart
Published: March 2003

The fate of the P1 plasmid of Escherichia coli was followed by time‐lapse photomicroscopy. A GFP–ParB fusion marked the plasmid during partition (segregation) to daughter cells at slow growth rate. The process differs from that previously inferred from statistical analysis of fixed cells. A focus of plasmid copies is captured at the cell centre. Immediately before cell division, the copies eject bidirectionally along the long axis of the cell. Cell division traps one or more plasmid copies in each daughter. They are not directed to a prescribed position but are free to move, associate and disassociate. Later, they are captured to the new cell centre to restart the cycle. A null P1 par mutant associates to form a focus, but it is neither captured nor ejected. A dominant negative ParB protein forms a plasmid focus that attaches to the cell centre but never ejects. It remains captive at the centre and blocks host cell division. The cells elongate. Eventually the intact focus is pushed to one side and the cells divide simultaneously in several places at the same time. This suggests that the wild‐type plasmid imposes a regulatory node on the host cell cycle, preventing cell division until its own segregation is completed.
The four different σ54 factors of Rhodobacter sphaeroides are not functionally interchangeable
Authors: Poggio Sebastian , Osorio Aurora , Dreyfus Georges , Camarena Laura
Published: March 2003

The σ54 factor is highly conserved in a large number of bacterial species. From the complete genome sequence of Rhodobacter sphaeroides, it was possible to identify four different sequences encoding potentially functional σ54 factors. In this work, we provide evidence that one of these copies (rpoN2) is specifically required to express the flagellar genes in this bacterium. A mutant strain carrying a lesion in the rpoN2 gene was unable to swim even though the RpoN1 and RpoN3 proteins were present in the cytoplasm. The possibility that the different copies of the σ54 factor might be specific for the transcription of a particular subset of σ54 promoters was reinforced by the fact that a mutant strain carrying a lesion in rpoN1 showed a severe growth defect in nitrogen‐free culture medium, even though the rpoN2 and rpoN4 genes were actively transcribed from a plasmid or from the chromosome. Different mech‐anisms that might be responsible for this specificity are discussed.
Characterization of a membrane‐linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes
Authors: Madec Edwige , Laszkiewicz Agnieszka , Iwanicki Adam , Obuchowski Michal , Séror Simone
Published: March 2003

PrkC was shown to be a eukaryotic‐like (Hanks‐type) protein kinase from Bacillus subtilis with a struc­‐tural organization similar to that of the eukaryotic sensor Ser/Thr or Tyr kinases (e.g. the TGF β or PDGF receptors). The molecule consists of a catalytic domain located in the cytoplasm, joined by a single transmembrane‐spanning region (TMD) to a large extracellular domain. Using a genetic reporter system, involving the cI repressor of lambda, evidence was obtained indicating that PrkC forms a dimer, involving both the TMD and the external domain in dimerization. The purified catalytic domain of PrkC was shown to autophosphorylate and to phosphorylate an external target, MBP, in both cases on threonine. These two functions require the completely conserved K40 residue in subdomain II, which is essential for enzymatic activity. Importantly, both the mutant deleted for prkC and a K40R mutant exhibit decreased efficiency of sporulation and a significant reduction in biofilm formation, demonstrating that the catalytic activity of PrkC is necessary for these two developmental processes. In addition, we showed that the product of prpC, a PPM phosphatase encoded by the adjacent gene, co‐transcribed with prkC, is also required for normal biofilm and spore formation.
Regulation and mode of action of the second small RNA activator of RpoS translation, RprA
Authors: Majdalani Nadim , Hernandez David , Gottesman Susan
Published: March 2003

Translation of the stationary phase sigma factor RpoS is stimulated by at least two small RNAs, DsrA and RprA. DsrA disrupts an inhibitory secondary structure in the rpoS leader mRNA by pairing with the upstream RNA. Mutations in rprA and compensating mutations in the rpoS leader demonstrate that RprA interacts with the same region of the RpoS leader as DsrA. This is the first example of two different small RNAs regulating a common target. Regulation of these RNAs differs. DsrA synthesis is increased at low temperature. We find that RprA synthesis is regulated by the RcsC/RcsB phosphorelay system, previously found to regulate capsule synthesis and promoters of ftsZ and osmC. An rcsB null mutation abolishes the basal level, whereas mutations in rcsC that activate capsule synthesis also activate expression of the rprA promoter. An essential site with similarity to other RcsB‐regulated promoters was defined in the rprA promoter. Activation of the RcsC/RcsB system leads to increased RpoS synthesis, in an RprA‐dependent fashion. This work suggests a new signal for RpoS translation and extends the global regulation effected by the RcsC/RcsB system to coregulation of RpoS with capsule and FtsZ.
Elongation factor Tu and E1 β subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae
Authors: Dallo Shatha F. , Kannan T. R. , Blaylock Mark W. , Baseman Joel B.
Published: March 2003

The interactions between pathogenic bacteria and extracellular matrix (ECM) components markedly influence the initiation and establishment of infection. We have identified two surface proteins of virulent Mycoplasma pneumoniae with molecular masses of 45 and 30 kDa that bind to the ECM constituent, fibronectin (Fn). These Fn‐binding proteins (FnBPs) were purified to near homogeneity using Fn‐coupled Sepharose 4B‐affinity column chromatography, and amino acid sequence analysis of the 45 and the 30 kDa proteins identified them as elongation factor Tu (EF‐Tu) and pyruvate dehydrogenase E1 β subunit (PDH‐B) respectively. The genes for EF‐Tu and PDH‐B were cloned, and the entire EF‐Tu gene and NH2‐terminus of PDH‐B (NPDH (pyruvate dehydrogenase E1 β subunit from amino acid 1–244)‐B) gene were overexpressed in Escherichia coli. The recombinant proteins, rEF‐Tu and rNPDH‐B, were purified to homogeneity by His‐tag affinity column chromatography and used to immunize rabbits. Purified rEF‐Tu and rNPDH‐B bound to Fn using a ligand immunoblot assay and ELISA. Immunogold electron microscopy with polyclonal antibodies reactive against rEF‐Tu (antirEF‐Tu) and rNPDH‐B (antirNPDH‐B) and whole cell radioimmunoprecipitation (WCRIP) revealed the surface location of these proteins. Adherence of viable M. pneumoniae to immobilized Fn was inhibited by antirEF‐Tu and antirNPDH‐B antisera in a dose‐dependent and cumulative manner. These results demonstrate that M. pneumoniae EF‐Tu and PDH‐B, in addition to their major cytoplasmic biosynthetic and metabolic roles, can be surface translocated, which confers additional important biological functions.
Rst1 and Rst2 are required for the a/α diploid cell type in yeast
Authors: Gelli Angie
Published: March 2003

In the budding yeast Saccharomyces cerevisiae, the preservation of the mating competent haploid (a or α) and the mating incompetent diploid (a/α) is necessary to prevent aneuploidy. Once haploid cells respond to pheromone, the mating‐specific signal transduction pathway is activated, and the MAP kinase Fus3 phosphorylates two specific repressor proteins Rst1 and Rst2 (also known as Dig1 and Dig2) to promote Ste12‐dependent transcription of mating‐specific genes. In contrast, diploid cells cannot mate because genes that encode components of the mating pathway are repressed through the combined action of the Mata1–Matα2 and Matα2–Mcm1 repressors. Surprisingly, repression of Ste12 by Rst1 and Rst2 is essential for diploid sterility. Homozygous deletion of both RST1 and RST2 (rst–) causes a/α diploid cells constitutively to express a‐specific genes and mate preferentially as a‐cells. This phenotype is sensitive to Ste12 dosage, as removal of one copy of STE12 completely reduces the ectopic activation of a‐specific genes. The Matα2–Mcm1 complex, which normally represses a‐specific genes, is defective in rst– diploids because Matα2 is destabilized in rst– diploids, possibly as a consequence of its relocalization from the nucleus to the cytoplasm. This study finds that Rst1 and Rst2 are necessary for the a/α diploid cell type. Rst1 and Rst2 are required in order to prevent the amplification of a robust Ste12 transcriptional programme that appears to over‐ride Matα2‐dependent repression of haploid and a‐specific genes.
Fibronectin mediates Opc‐dependent internalization of Neisseria meningitidis in human brain microvascular endothelial cells
Authors: Unkmeir Alexandra , Latsch Kirsten , Dietrich Guido , Wintermeyer Eva , Schinke Birgitta , Schwender Stefan , Kim Kwang Sik , Eigenthaler Martin , Frosch Matthias
Published: March 2003

A central step in the pathogenesis of bacterial meningitis caused by Neisseria meningitidis (the meningococcus) is the interaction of the bacteria with cells of the blood–brain barrier. In the present study, we analysed the invasive potential of two strains representing hypervirulent meningococcal lineages of the ET‐5 and ET‐37 complex in human brain‐derived endothelial cells (HBEMCs). In contrast to previous observations made with epithelial cells and human umbilical vein‐derived endothelial cells (HUVECs), significant internalization of encapsulated meningococci by HBMECs was observed. However, this uptake was found only for the ET‐5 complex isolate MC58, and not for an ET‐37 complex strain. Furthermore, the uptake of meningococci by HBMECs depended on the presence of human serum, whereas serum of bovine origin did not promote the internalization of meningococci in HBMECs. By mutagenesis experiments, we demonstrate that internalization depended on the expression of the opc gene, which is present in meningococci of the ET‐5 complex, but absent in ET‐37 complex meningococci. Chromatographic separation of human serum proteins revealed fibronectin as the uptake‐promoting serum factor, which binds to HBMECs via α5β1 integrin receptors. These data provide evidence for unique molecular mechanisms of the interaction of meningococci with endothelial cells of the blood–brain barrier and contribute to our understanding of the pathogenesis of meningitis caused by meningococci of different clonal lineages.
Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti
Authors: Pym Alexander S. , Brodin Priscille , Brosch Roland , Huerre Michel , Cole Stewart T.
Published: March 2003

Although large human populations have been safely immunized against tuberculosis with two live vaccines, Mycobacterium bovis BCG or Mycobacterium microti, the vole bacillus, the molecular basis for the avirulence of these vaccine strains remains unknown. Comparative genomics has identified a series of chromosomal deletions common to both virulent and avirulent species but only a single locus, RD1, that has been deleted from M. bovis BCG and M. microti. Restoration of RD1, by gene knock‐in, resulted in a marked change in colonial morphology towards that of virulent tubercle bacilli. Three RD1‐encoded proteins were localized in the cell wall, and two of them, the immunodominant T‐cell antigens ESAT‐6 and CFP‐10, were also found in culture supernatants. The BCG::RD1 and M. microti::RD1 knock‐ins grew more vigorously than controls in immunodeficient mice, inducing extensive splenomegaly and granuloma formation. Increased persistence and partial reversal of attenuation were observed when immunocompetent mice were infected with the BCG::RD1 knock‐in, whereas BCG controls were cleared. Knocking‐in five other RD loci did not affect the virulence of BCG. This study describes a genetic lesion that contributes to safety and opens new avenues for vaccine development.
Integron‐encoded IntI integrases preferentially recognize the adjacent cognate attI site in recombination with a 59‐be site
Authors: Collis Christina M. , Kim Mi‐Jurng , Stokes H. W. , Hall Ruth M.
Published: March 2003

Integrons have the capacity to capture small mobile elements known as gene cassettes, and this reaction is catalysed by integron‐encoded IntI integrases. IntI integrases form a distinct family within the tyrosine recombinase superfamily and include a characteristic additional domain that is well conserved. Two different IntI enzymes were used to examine their ability to recognize heterologous attI sites in both integration and excision assays. IntI1 and IntI3 are 59% identical and catalyse both integrative and excisive recombination between a cassette‐associated 59‐be site and the cognate attI1 or attI3 site. Integrative recombination events involving a 59‐be and a non‐cognate attI site, attI2 and attI3 for IntI1 or attI1 and attI2 for IntI3, were detected extremely rarely. In cassette excision assays, the non‐cognate attI3 site was recognized by IntI1, but attI1 was not well recognized by IntI3. The purified IntI1 and IntI3 proteins bound strongly only to their cognate attI site.
Intestinal short‐chain fatty acids alter Salmonella typhimurium invasion gene expression and virulence through BarA/SirA
Authors: Lawhon Sara D. , Maurer Russell , Suyemoto Mitsu , Altier Craig
Published: March 2003

Salmonella typhimurium causes enteric and systemic disease by invading the intestinal epithelium of the distal ileum, a process requiring the invasion genes of Salmonella pathogenicity island 1 (SPI‐1). BarA, a sensor kinase postulated to interact with the response regulator SirA, is required for the expression of SPI‐1 invasion genes. We found, however, that a barA null mutation had little effect on virulence using the mouse model for septicaemia. This confounding result led us to seek environmental signals present in the distal ileum that might supplant the need for BarA. We found that acetate restored the expression of invasion genes in the barA mutant, but had no effect on a sirA mutant. Acetate had its effect only at a pH that allowed its accumulation within the bacterial cytoplasm and not with the deletion of ackA and pta, the two genes required to produce acetyl‐phosphate. These results suggest that the rising concentration of acetate in the distal ileum provides a signal for invasion gene expression by the production of acetyl‐phosphate in the bacterial cytoplasm, a pathway that bypasses barA. We also found that a Δ(ackA–pta) mutation alone had no effect on virulence but, in combination with Δ(barA), it increased the oral LD50 24‐fold. Thus, the combined loss of the BarA‐ and acetate‐dependent pathways is required to reduce virulence. Two other short‐chain fatty acids (SCFA), propionate and butyrate, present in high concentrations in the caecum and colon, had effects opposite to those of acetate: neither restored invasion gene expression in the barA mutant, and both, in fact, reduced expression in the wild‐type strain. Further, a combination of SCFAs found in the distal ileum restored invasion gene expression in the barA mutant, whereas colonic conditions failed to do so and also reduced expression in the wild‐type strain. These results suggest that the concentration and composition of SCFAs in the distal ileum provide a signal for productive infection by Salmonella, whereas those of the large intestine inhibit invasion.
Ectopic RNase E sites promote bypass of 5′‐end‐dependent mRNA decay in Escherichia coli
Authors: Baker Kristian E. , Mackie George A.
Published: March 2003

In Escherichia coli, 5′‐terminal stem–loops form major impediments to mRNA decay, yet conditions that determine their effectiveness or the use of alternative decay pathway(s) are unclear. A synthetic 5′‐terminal hairpin stabilizes the rpsT mRNA sixfold. This stabilization is dependent on efficient translational initiation and ribosome transit through at least two‐thirds of the coding sequence past a major RNase E cleavage site in the rpsT mRNA. Insertion of a 12–15 residue ‘ectopic’ RNase E cleavage site from either the rne leader or 9S pre‐rRNA into the 5′‐non‐coding region of the rpsT mRNA significantly reduces the stabilizing effect of the terminal stem–loop, dependent on RNase E. A similar insertion into the rpsT coding sequence is partially destabilizing. These findings demonstrate that RNase E can bypass an interaction with the 5′‐terminus, and exploit an alternative ‘internal entry’ pathway. We propose a model for degradation of the rpsT mRNA, which explains the hierarchy of protection afforded by different 5′‐termini, the use of internal entry for bypass of barriers to decay, ‘ectopic sites’ and the role of translating ribosomes.
Copper‐mediated reversal of defective laccase in a Δvph1 avirulent mutant of Cryptococcus neoformans
Authors: Zhu Xudong , Gibbons Jack , Zhang Shirong , Williamson Peter R.
Published: March 2003

Previous studies have shown that a Δvph1 Cryptococcus neoformans mutant defective in vesicular acidification lacked several important virulence factors including a copper‐containing laccase and was avirulent in a mouse model. In the present studies, we characterized laccase transcription and protein production to obtain insights into the mechanism of the vph1 mutation in this pathogen. Although transcription and protein expression were somewhat reduced, laccase protein was found to be successfully translated and correctly targeted to the cell wall in the Δvph1 mutant as shown by Western blot and immuno‐electron microscopy, despite a complete lack of laccase activity. Laccase activity was substantially restored in metabolically active Δvph1 cells at 30°C by addition of 100 µM copper sulphate. This restoration by copper was found to occur through both transcriptional and post‐translational mechanisms. Laccase transcriptional induction by copper was found to be dependent on enhancer region II within the 5′‐untranslated region of CNLAC1. Copper was also found to restore partial activity to Δvph1 cells at 0°C, suggesting that cell wall laccase was expressed in the mutant as an apo‐enzyme. Apo‐laccase restoration by copper was found to be facilitated by an acidic environment, consistent with a role for the vacuolar (H+)‐ATPase proton pump in copper assembly of laccase in C. neoformans.
Transcriptional organization and regulation of the Escherichia coli K30 group 1 capsule biosynthesis (cps) gene cluster
Authors: Rahn Andrea , Whitfield Chris
Published: March 2003

Escherichia coli
group 1 capsules are important virulence determinants, yet little is known about the transcriptional organization or regulation of their biosynthetic (
cps
) operons. Transcription of the prototype serotype K30 cluster is modulated by the JUMPStart–RfaH antitermination mechanism, with the
cps
promoter being localized to a region immediately upstream of the JUMPStart sequence. A putative stem–loop structure located within the K30
cps
cluster separates conserved genes with products that are required for surface expression of capsule from serotype‐specific genes encoding enzymes for polymer repeat‐unit synthesis and polymerization. This putative stem–loop structure significantly reduces transcription in a termination‐probe vector and may contribute to differential expression of the
cps
genes. Previous work indicated that increased amounts of group 1 capsular polysaccharide synthesis resulted from the overexpression of the Rcs (
r
egulator of
c
apsule
s
ynthesis) proteins. However, neither overexpression of the transcriptional activator RcsB nor an
rcsB
::
aadA
chromosomal insertion altered the level of transcription measured by
cps::lacZ
fusions. In the group 1 strains examined, an RcsAB box was found immediately upstream of
galF
, a gene involved in the production of sugar nucleotide precursors. Overexpression of RcsB was found to result in a threefold increase in transcription of a
galF::lacZ
chromosomal fusion. Moreover, overexpression of GalF gave rise to a two‐ to threefold increase in cell‐free as well as cell‐associated capsule, without affecting
cps::lacZ
activity. These results indicate that transcription of the
E. coli
group 1 capsule cluster itself is not regulated by the Rcs system and may, in fact, be constitutive. However, the Rcs system can potentially influence levels of capsular polysaccharide production by increasing
galF transcription and influencing the available pool of biosynthetic precursors.