bluuebunny/crossref_metadata_2025_split huggingface dataset:
MexAB‐OprM hyperexpression in NalC‐type multidrug‐resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720‐PA3719
Authors: Cao Lily , Srikumar Ramakrishnan , Poole Keith 
Published: August 2004

SummaryMexAB‐OprM is a multidrug efflux system that contributes to intrinsic and acquired multidrug resistance in Pseudomonas aeruginosa, the latter as a result of mutational hyperexpression of the mexAB‐oprM operon. While efflux gene hyperexpression typically results from mutations in the linked mexR repressor gene, it also occurs independently of mexR mutations in so‐called nalC mutants that demonstrate more modest mexAB‐oprM expression and, thus, more modest multidrug resistance than do mexR strains. Using a transposon insertion mutagenesis approach, nalC mutant strains were selected and the disrupted gene, PA3721, identified. Amplification and sequencing of this gene from previously isolated spontaneous nalC mutants revealed the presence of mutations in all instances and as such, PA3721 has been renamed nalC. PA3721 (nalC) encodes a probable repressor of the TetR/AcrR family and occurs upstream of an apparent two‐gene operon, PA3720–PA3719, whose expression was negatively regulated by PA3721. Thus, PA3720‐PA3719 was hyperexpressed in transposon insertion and spontaneous nalC mutants. The loss of PA3719 but not of PA3720 expression in a spontaneous nalC mutant reduced MexAB‐OprM expression to wild‐type levels and compromised multidrug resistance, an indication that hyperexpression of PA3719 only was necessary for the nalC phenotype. Introduction of PA3719 into wild‐type P. aeruginosa on a multicopy plasmid was, in fact, sufficient to promote elevated MexAB‐OprM expression and multidrug resistance characteristic of a nalC strain. Thus, the nalC (PA3721) mutation serves only to enhance PA3720‐PA3719 expression, with expression of PA3719 (encodes a 53 amino acid protein of predicted pI 10.4) directly or indirectly impacting MexAB‐OprM expression. Intriguingly, nalC strains produce markedly elevated levels of stable MexR protein suggesting that PA3720‐PA3719 hyperexpression somehow modulates MexR repressor activity. The deduced products of PA3720‐PA3719 show no homology to sequences presently in the GenBank databases, however, and as such provide no clues as to how this might occur.
Repression of the RcsC‐YojN‐RcsB phosphorelay by the IgaA protein is a requisite for Salmonella virulence
Authors: Domínguez‐Bernal Gustavo , Pucciarelli M. Graciela , Ramos‐Morales Francisco , García‐Quintanilla Meritxell , Cano David A. , Casadesús Josep , García‐del Portillo Francisco 
Published: August 2004

SummaryBacterial pathogenesis relies on regulators that activate virulence genes. Some of them act, in addition, as repressors of specific genes. Intracellular‐growth‐attenuator‐A (IgaA) is a Salmonella enterica membrane protein that prevents overactivation of the RcsC‐YojN‐RcsB regulatory system. This negative control is critical for growth because disruption of the igaA gene is only possible in rcsC, yojN or rcsB strains. In this work, we examined the contribution of this regulatory circuit to virulence. Viable igaA point mutant alleles were isolated and characterized. These alleles encode IgaA variants leading to different levels of activation of the RcsC‐YojN‐RcsB system. IgaA‐mediated repression of the RcsB‐YojN‐RcsC system occurred at the post‐translational level, as shown by chromosomal epitope tagging of the rcsC, yojN and rcsB genes. The activity of the RcsC‐YojN‐RcsB system, monitored with the product of a tagged gmd‐3xFLAG gene (positively regulated by RcsC‐YojN‐RcsB), was totally abolished by wild‐type bacteria in mouse target organs. Such tight repression occurred only in vivo and was mediated by IgaA. Shutdown of the RcsC‐YojN‐RcsB system is a requisite for Salmonella virulence since all igaA point mutant strains were highly attenuated. The degree of attenuation correlated to that of the activation status of RcsC‐YojN‐RcsB. In some cases, the attenuation recorded was unprecedented, with competitive index (CI) values as low as 10−6. Strikingly, IgaA is a protein absolutely dispensable for virulence in mutant strains having a non‐functional RcsC‐YojN‐RcsB system. To our knowledge, IgaA exemplifies the first protein that contributes to virulence by exclusively acting as a negative regulator upon host colonization.
Regulatory networks affected by iron availability in Candida albicans
Authors: Lan Chung‐Yu , Rodarte Gabriel , Murillo Luis A. , Jones Ted , Davis Ronald W. , Dungan Jan , Newport George , Agabian Nina 
Published: August 2004

SummaryIron, an essential element for almost every organism, serves as a regulatory signal for the expression of virulence determinants in many prokaryotic and eukaryotic pathogens. Using a custom Affymetrix GeneChip© representing the entire Candida albicans genome, we examined the changes in genome‐wide gene expression in this opportunistic pathogen as a function of alterations in environmental concentrations of iron. A total of 526 open reading frame (ORF) transcripts are more highly expressed when the levels of available iron are low, while 626 ORF transcripts are more highly expressed in high‐iron conditions. The transcripts dominantly affected by iron concentration range from those associated with cell‐surface properties to others which affect mitochondrial function, iron transport and virulence‐related secreted hydrolases. Moreover gene expression as assayed in DNA microarrays confirms and extends reports of alterations in cell‐surface antigens and drug sensitivity correlated with iron availability. To understand how these genes and pathways might be regulated, we isolated a gene designated SFU1 that encodes a homologue of the Ustilago maydis URBS1, a transcriptional repressor of siderophore uptake/biosynthesis. Comparisons between wild‐type and SFU1‐null mutant strains revealed 139 potential target genes of Sfu1p; many of which are iron‐responsive. Together, these results not only expand our understanding of global iron regulation in C. albicans, but also provide insights into the potential role of iron availability in C. albicans virulence.
The sirodesmin biosynthetic gene cluster of the plant pathogenic fungus Leptosphaeria maculans
Authors: Gardiner Donald M. , Cozijnsen Anton J. , Wilson Leanne M. , Pedras M. Soledade C. , Howlett Barbara J. 
Published: August 2004

SummarySirodesmin PL is a phytotoxin produced by the fungus Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). This phytotoxin belongs to the epipolythiodioxopiperazine (ETP) class of toxins produced by fungi including mammalian and plant pathogens. We report the cloning of a cluster of genes with predicted roles in the biosynthesis of sirodesmin PL and show via gene disruption that one of these genes (encoding a two‐module non‐ribosomal peptide synthetase) is essential for sirodesmin PL biosynthesis. Of the nine genes in the cluster tested, all are co‐regulated with the production of sirodesmin PL in culture. A similar cluster is present in the genome of the opportunistic human pathogen Aspergillus fumigatus and is most likely responsible for the production of gliotoxin, which is also an ETP. Homologues of the genes in the cluster were also identified in expressed sequence tags of the ETP producing fungus Chaetomium globosum. Two other fungi with publicly available genome sequences, Magnaporthe grisea and Fusarium graminearum, had similar gene clusters. A comparative analysis of all four clusters is presented. This is the first report of the genes responsible for the biosynthesis of an ETP.
Dimerization properties of TraM, the antiactivator that modulates TraR‐mediated quorum‐dependent expression of the Ti plasmid tra genes
Authors: Qin Yinping , Smyth Audra J. , Su Shengchang , Farrand Stephen K. 
Published: August 2004

SummaryTraM, an 11.2 kDa antiactivator, modulates the acyl‐homoserine lactone‐mediated autoinduction of Ti plasmid conjugative transfer by interacting directly with TraR, the quorum‐sensing transcriptional activator. Most antiactivators and antisigma factors examined to date act in dimer form. However, whether, and if so, how TraM dimerizes is unknown. Analyses based on a genetic assay using fusions of TraM to the λ cI DNA binding domain, and biochemical assays using chemical crosslinking and gel filtration chromatography showed that TraM forms homodimers. Although SDS‐PAGE studies suggested that the lone cysteine residue at position 71 was involved in interprotomer disulfide‐bridging in TraM, altering Cys‐71 to a serine did not significantly affect dimerization or the antiactivator activity of this mutant protein when expressed at wild‐type levels in vivo. Analysis of N‐terminal, C‐terminal, and internal deletion mutants of TraM identified two regions of the protein involved in dimerization; one located within a segment between residues 20 and 50, and the other located to a segment between residues 67 and 96. Both regions are required for formation of fully stable dimers. Analysis of the activity of these deletion mutants in vivo, and their ability to bind TraR and to disrupt TraR‐DNA complexes in vitro, suggests that while the internal segment of the protein is required for dimerization, determinants located at the far C‐terminus and beginning at between residues 10 and 20 at the N‐terminus play a role in TraR binding and antiactivator function. When co‐expressed with λ cI′::TraR fusions, wild‐type TraM mediated quormone‐independent dimerization of the transcriptional activator, suggesting that dimers of TraM can multimerize TraR.
A novel ERK‐like, CRK‐like protein kinase that modulates growth in Trypanosoma brucei via an autoregulatory C‐terminal extension
Authors: Ellis James , Sarkar Mitali , Hendriks Edward , Matthews Keith 
Published: August 2004

SummaryThe protozoan parasite Trypanosoma brucei undergoes a complex developmental cycle coordinated with cell cycle control. These processes in eukaryotes are frequently regulated through mitogen‐activated protein kinases (MAPKs) and cyclin‐dependent protein kinases (CDKs), respectively. We have discovered a novel protein kinase which shares features of both ERK‐type MAPKs and CDKs (T. brucei ERK‐like, CDK‐like protein kinase). This molecule, named TbECK1, is similar to the unusual mammalian KKIAMRE protein kinase family. Moreover, TbECK1 possesses a long C‐terminal extension reminiscent of those found in mammalian ERK5, ERK7 and ERK8. Expression analyses demonstrate that TbECK1 is constitutively expressed during the trypanosome life cycle at both RNA and protein level. In transgenic parasites we demonstrate that expression of a mutant of TbECK1 that lacks the C‐terminal extension produces a slow growth phenotype, associated with the appearance of cells with aberrant karyotypes. Using this as an assay we further demonstrate that the phenotype is dependent upon the potential for catalytic activity of TbECK1 and on the integrity of at least one of the phosphorylable amino acids in its phosphorylation lip. C‐terminal extensions are a common feature of kinetoplastid protein kinases. Our results demonstrate for the first time that this domain has a regulatory function.
Tyrosine site‐specific recombinases mediate DNA inversions affecting the expression of outer surface proteins of Bacteroides fragilis
Authors: Weinacht Katja G. , Roche Hazeline , Krinos Corinna M. , Coyne Michael J. , Parkhill Julian , Comstock Laurie E. 
Published: August 2004

SummaryThe chromosome of Bacteroides fragilis has been shown to undergo 13 distinct DNA inversions affecting the expression of capsular polysaccharides and mediated by a serine site‐specific recombinase designated Mpi. In this study, we demonstrate that members of the tyrosine site‐specific recombinase family, conserved in B. fragilis, mediate additional DNA inversions of the B. fragilis genome. These DNA invertases flip promoter regions in their immediate downstream region. The genetic organization of the genes regulated by these invertible promoter regions suggests that they are operons and many of the products are predicted to be outer membrane proteins. Phenotypic analysis of a deletion mutant of one of these DNA invertases, tsr15 (aapI), which resulted in the promoter region for the downstream genes being locked ON, confirmed the synthesis of multiple surface proteins by this operon. In addition, this deletion mutant demonstrated an autoaggregative phenotype and showed significantly greater adherence than wild‐type organisms in a biofilm assay, suggesting a possible functional role for these phase‐variable outer surface proteins. This study demonstrates that DNA inversion is a universal mechanism used by this commensal microorganism to phase vary expression of its surface molecules and involves at least three conserved DNA invertases from two evolutionarily distinct families.
Analysis of the Frz signal transduction system of Myxococcus xanthus shows the importance of the conserved C‐terminal region of the cytoplasmic chemoreceptor FrzCD in sensing signals
Authors: Bustamante Víctor H. , Martínez‐Flores Irma , Vlamakis Hera C. , Zusman David R. 
Published: August 2004

SummaryThe Frz chemosensory system controls directed motility in Myxococcus xanthus by regulating cellular reversal frequency. M. xanthus requires the Frz system for vegetative swarming on rich media and for cellular aggregation during fruiting body formation on starvation media. The Frz signal transduction pathway is formed by proteins that share homology with chemotaxis proteins from enteric bacteria, which are encoded in the frzA‐F putative operon and the divergently transcribed frzZ gene. FrzCD, the Frz system chemoreceptor, contains a conserved C‐terminal module present in methyl‐accepting chemotaxis proteins (MCPs); but, in contrast to most MCPs, FrzCD is localized in the cytoplasm and the N‐terminal region of FrzCD does not contain transmembrane or sensing domains, or even a linker region. Previous work on the Frz system was limited by the unavailability of deletion strains. To understand better how the Frz system functions, we generated a series of in‐frame deletions in each of the frz genes as well as regions encoding the N‐terminal portion of FrzCD. Analysis of mutants containing these deletions showed that FrzCD (MCP), FrzA (CheW) and FrzE (CheA–CheY) control vegetative swarming, responses to repellents and directed movement during development, thus constituting the core components of the Frz pathway. FrzB (CheW), FrzF (CheR), FrzG (CheB) and FrzZ (CheY–CheY) are required for some but not all responses. Furthermore, deletion of ≈ 25 amino acids from either end of the conserved C‐terminal region of FrzCD results in a constitutive signalling state of FrzCD, which induces hyper‐reversals with no net cell movement. Surprisingly, deletion of the N‐terminal region of FrzCD shows only minor defects in swarming. Thus, signal input to the Frz system must be sensed by the conserved C‐terminal module of FrzCD and not the usual N‐terminal region. These results indicate an alternative mechanism for signal sensing with this cytoplasmic MCP.
Synthesis of group A streptococcal virulence factors is controlled by a regulatory RNA molecule
Authors: Mangold Monika , Siller Maria , Roppenser Bernhard , Vlaminckx Bart J. M. , Penfound Tom A. , Klein Reinhard , Novak Rodger , Novick Richard P. , Charpentier Emmanuelle 
Published: August 2004

SummaryThe capacity of pathogens to cause disease depends strictly on the regulated expression of their virulence factors. In this study, we demonstrate that the untranslated mRNA of the recently described streptococcal pleiotropic effect locus (pel), which incidentally contains sagA, the structural gene for streptolysin S, is an effector of virulence factor expression in group A beta‐haemolytic streptococci (GAS). Our data suggest that the regulation by pel RNA occurs at both transcriptional (e.g. emm, sic, nga) and post‐transcriptional (e.g. SpeB) levels. We could exclude the possibility that the pel phenotype was linked to a polar effect on downstream genes (sagB‐I). Remarkably, the RNA effector is regulated in a growth phase‐dependent fashion and we provide evidence that pel RNA expression is induced by conditioned media.
Measurement of SOS expression in individual Escherichia coli K‐12 cells using fluorescence microscopy
Authors: McCool Jesse D. , Long Edward , Petrosino Joseph F. , Sandler Hilary A. , Rosenberg Susan M. , Sandler Steven J. 
Published: August 2004

SummaryMany recombination, DNA repair and DNA replication mutants have high basal levels of SOS expression as determined by a sulAp‐lacZ reporter gene system on a population of cells. Two opposing models to explain how the SOS expression is distributed in these cells are: (i) the ‘Uniform Expression Model (UEM)’ where expression is evenly distributed in all cells or (ii) the ‘Two Population Model (TPM)’ where some cells are highly induced while others are not at all. To distinguish between these two models, a method to quantify SOS expression in individual bacterial cells was developed by fusing an SOS promoter (sulAp) to the green fluorescent protein (gfp) reporter gene and inserting it at attλ on the Escherichia coli chromosome. It is shown that the fluorescence in sulAp‐gfp cells is regulated by RecA and LexA. This system was then used to distinguish between the two models for several mutants. The patterns displayed by priA, dnaT, recG, uvrD, dam, ftsK, rnhA, polA and xerC mutants were explained best by the TPM while only lexA (def), lexA3 (ind–) and recA defective mutants were explained best by the UEM. These results are discussed in a context of how the processes of DNA replication and recombination may affect cells in a population differentially.
Shear‐dependent ‘stick‐and‐roll’ adhesion of type 1 fimbriated Escherichia coli
Authors: Thomas Wendy E. , Nilsson Lina M. , Forero Manu , Sokurenko Evgeni V. , Vogel Viola 
Published: August 2004

SummaryIt is generally assumed that bacteria are washed off surfaces as fluid flow increases because they adhere through ‘slip‐bonds’ that weaken under mechanical force. However, we show here that the opposite is true for Escherichia coli attachment to monomannose‐coated surfaces via the type 1 fimbrial adhesive subunit, FimH. Raising the shear stress (within the physiologically relevant range) increased accumulation of type 1 fimbriated bacteria on monomannose surfaces by up to two orders of magnitude, and reducing the shear stress caused them to detach. In contrast, bacterial binding to anti‐FimH antibody‐coated surfaces showed essentially the opposite behaviour, detaching when the shear stress was increased. These results can be explained if FimH is force‐activated; that is, that FimH mediates ‘catch‐bonds’ with mannose that are strengthened by tensile mechanical force. As a result, on monomannose‐coated surfaces, bacteria displayed a complex ‘stick‐and‐roll’ adhesion in which they tended to roll over the surface at low shear but increasingly halted to stick firmly as the shear was increased. Mutations in FimH that were predicted earlier to increase or decrease force‐induced conformational changes in FimH were furthermore shown here to increase or decrease the probability that bacteria exhibited the stationary versus the rolling mode of adhesion. This ‘stick‐and‐roll’ adhesion could allow type 1 fimbriated bacteria to move along mannosylated surfaces under relatively low flow conditions and to accumulate preferentially in high shear regions.
Role of two essential domains of Escherichia coli FtsA in localization and progression of the division ring
Authors: Rico Ana Isabel , García‐Ovalle Marta , Mingorance Jesús , Vicente Miguel 
Published: August 2004

SummaryThe FtsA protein is a member of the actin superfamily that localizes to the bacterial septal ring during cell division. Deletions of domain 1C or the S12 and S13 β‐strands in domain 2B of the Escherichia coli FtsA, previously postulated to be involved in dimerization, result in partially active proteins that do not allow the normal progression of septation. The truncated FtsA protein lacking domain 1C (FtsAΔ1C) localizes in correctly placed division rings, together with FtsZ and ZipA, but does not interact with other FtsA molecules in the yeast two‐hybrid assay, and fails to recruit FtsQ and FtsN into the division ring. The rings containing FtsAΔ1C are therefore incomplete and do not support division. The production of high levels of FtsAΔ1C causes filamentation, an effect that has been reported to result as well from the imbalance between FtsA+ and FtsZ+ molecules. These data indicate that the domain 1C of FtsA participates in the interaction of the protein with other FtsA molecules and with the other proteins that are incorporated at later stages of ring assembly, and is not involved in the interaction with FtsZ and the localization of FtsA to the septal ring. The deletion of the S12–S13 strands of domain 2B generates a protein (FtsAΔS12–13) that retains the ability to interact with FtsA+. When the mutated protein is expressed at wild‐type levels, it localizes into division rings and recruits FtsQ and FtsN, but it fails to sustain septation at normal levels resulting in filamentation. A fivefold overexpression of FtsAΔS12–13 produces short cells that have normal division rings, but also cells with polar localization of the mutated protein, and cells with rings at abnormal positions that result in the production of a fraction (15%) of small nucleoid‐free cells. The S12–S13 strands of domain 2B are not essential for septation, but affect the localization of the division ring.
Aggressiveness and host specificity of Brazilian isolates of Phytophthora infestans
Authors: Suassuna N. D. , Maffia L. A. , Mizubuti E. S. G. 
Published: August 2004

The population of Phytophthora infestans in Brazil consists of two clonal lineages, US‐1 associated with tomatoes and BR‐1 associated with potatoes. To assess whether host specificity in these lineages resulted from differences in aggressiveness to potato and tomato, six aggressiveness‐related epidemiological components – infection frequency (IF), incubation period (IP), latent period (LP), lesion area (LA), lesion expansion rate (LER) and sporulation at several lesion ages (SSLA) – were measured on detached leaflets of late blight‐susceptible potato and tomato plants. Infection frequency of US‐1 was similar on potato and tomato leaflets, but IF of BR‐1 was somewhat reduced on tomato. Incubation period was longer on both hosts with US‐1, although this apparent lineage affect was not significant. Overall there was no host effect on IP. On potato, BR‐1 had a shorter LP (110·3 h) and a larger LA (6·5 cm2) than US‐1 (LP = 162·0 h; LA = 2·8 cm2). The highest LER resulted when isolates of BR‐1 (0·121 cm2 h−1) and US‐1 (0·053 cm2 h−1) were inoculated on potato and tomato leaflets, respectively. The highest values of the area under the sporulation capacity curve (AUSC) were obtained for isolates of US‐1 inoculated on tomato leaflets (6146) and for isolates of BR‐1 on potato leaflets (3775). In general, higher values of LA, LER, SSLA and AUSC, and shorter values of LP were measured when isolates of a clonal lineage were inoculated on their original host than with the opposite combinations. There is evidence that there are quantitative differences in aggressiveness components between isolates of US‐1 and BR‐1 clonal lineages that probably contribute to host specificity of P. infestans populations in Brazil.