bluuebunny/crossref_metadata_2025 huggingface dataset:
The cmaR gene of Corynebacterium ammoniagenes performs a novel regulatory role in the metabolism of sulfur-containing amino acids
Authors: Lee Seok-Myung , Hwang Byung-Joon , Kim Younhee , Lee Heung-Shick
Published: June 2009

A novel regulatory gene, which performs an essential function in sulfur metabolism, has been identified inCorynebacterium ammoniagenesand was designatedcmaR(cysteine andmethionine regulator inC.ammoniagenes). ThecmaR-disrupted strain (ΔcmaR) lost the ability to grow on minimal medium, and was identified as a methionine and cysteine double auxotroph. The mutant strain proved unable to convert cysteine to methionine (and vice versa), and lost the ability to assimilate and reduce sulfate to sulfide. In the ΔcmaRstrain, the mRNAs of the methionine biosynthetic genesmetYX,metBandmetFEwere significantly reduced, and the activities of the methionine biosynthetic enzymes cystathionineγ-synthase,O-acetylhomoserine sulfhydrylase, and cystathionineβ-lyase were relatively low, thereby suggesting that thecmaRgene exerts a positive regulatory effect on methionine biosynthetic genes. In addition, with the exception ofcysK, reduced transcription levels of the sulfur-assimilatory genescysIXYZandcysHDNwere noted in thecmaR-disrupted strain, which suggests that sulfur assimilation is also under the positive control of thecmaRgene. Furthermore, the expression of thecmaRgene itself was strongly induced via the addition of cysteine or methionine alone, but not the introduction of both amino acids together to the growth medium. In addition, the expression of thecmaRgene was enhanced in anmcbR-disrupted strain, which suggests thatcmaRis under the negative control of McbR, which has been identified as a global regulator of sulfur metabolism. DNA binding of the purified CmaR protein to the promoter region of its target genes could be demonstratedin vitro. No metabolite effector was required for the protein to bind DNA. These results demonstrated that thecmaRgene ofC. ammoniagenesplays a role similar to but distinct from that of the functional homologuecysRofCorynebacterium glutamicum.
Characterization and subcellular localization of a bacterial flotillin homologue
Authors: Donovan Catriona , Bramkamp Marc
Published: June 2009

The process of endospore formation in Bacillus subtilis is complex, requiring the generation of two distinct cell types, a forespore and larger mother cell. The development of these cell types is controlled and regulated by cell type-specific gene expression, activated by a σ-factor cascade. Activation of these cell type-specific sigma factors is coupled with the completion of polar septation. Here, we describe a novel protein, YuaG, a eukaryotic reggie/flotillin homologue that is involved in the early stages of sporulation of the Gram-positive model organism B. subtilis. YuaG localizes in discrete foci in the membrane and is highly dynamic. Purification of detergent-resistant membranes revealed that YuaG is associated with negatively charged phospholipids, e.g. phosphatidylglycerol (PG) or cardiolipin (CL). However, localization of YuaG is not always dependent on PG/CL in vivo. A yuaG disruption strain shows a delay in the onset of sporulation along with reduced sporulation efficiency, where the spores develop to a certain stage and then appear to be trapped at this stage. Our results indicate that YuaG is involved in the early stage of spore development, probably playing a role in the signalling cascade at the onset of sporulation.
Population heterogeneity in Methylobacterium extorquens AM1
Authors: Strovas Tim J. , Lidstrom Mary E.
Published: June 2009

Heterogeneity of cells within exponentially growing populations was addressed in a bacterium, the facultative methylotroph Methylobacterium extorquens AM1. A transcriptional fusion between a well-characterized methanol-inducible promoter (PmxaF
) and gfpuv
was used with flow cytometry to analyse the distribution of gene expression in populations grown on either succinate or methanol, correlated with forward scatter as a measure of cell size. These cell populations were found to consist of three major subpopulations defined by cells that were actively growing and dividing, newly divided, and non-dividing. Through the use of flow cytometry, it was demonstrated that a significant percentage of the total population did not respond to carbon shift. In addition, these experiments demonstrated that a small subset of the total population was significantly brighter than the rest of the population and dominated fluorimetry data. These results were corroborated with a continuous flow-through system and laser scanning microscopy, confirming that subpopulations, not discernible in the population average, dominate population response. These results demonstrate that the combination of flow cytometry and microscopic single-cell analysis can be effectively used to determine the dynamics of subpopulations in population response. In addition, they support the concept that physiological diversity in isogenic populations can poise some proportion of the population to respond appropriately to changing conditions.
Survival of uropathogenic Escherichia coli in the murine urinary tract is dependent on OmpR
Authors: Schwan William R.
Published: June 2009

UropathogenicEscherichia coli(UPEC) can grow in environments with significantly elevated osmolarities, such as murine and human urinary tracts. OmpR is the response regulator part of a two-component OmpR–EnvZ regulatory system that responds to osmotic stresses. To determine the role of OmpR in UPEC survival, a ΔompRmutant was created in the UPEC clinical isolate NU149. The ΔompRmutant had a growth defect compared with the wild-type strain under osmotic stress conditions; this defect was complemented by the full-lengthompRgene on a plasmid, but not with a mutant OmpR with an alanine substitution for aspartic acid at the phosphorylation site at position 55. Furthermore, the ΔompRmutant displayed up to 2-log reduction in bacterial cell numbers in murine bladders and kidneys compared with wild-type bacteria after 5 days of infection. The ability of the bacteria to survive was restored to wild-type levels when the ΔompRmutant strain was complemented with wild-typeompR, but not when the alanine-substitutedompRgene was used. This study has fulfilled molecular Koch's postulates by showing the pivotal role OmpR plays in UPEC survival within the murine urinary tract.
Tellurite-mediated disabling of [4Fe–4S] clusters of Escherichia coli dehydratases
Authors: Calderón Iván L. , Elías Alex O. , Fuentes Eugenia L. , Pradenas Gonzalo A. , Castro Miguel E. , Arenas Felipe A. , Pérez José M. , Vásquez Claudio C.
Published: June 2009

The tellurium oxyanion tellurite is toxic for most organisms and it seems to alter a number of intracellular targets. In this work the toxic effects of tellurite uponEscherichia coli[4Fe–4S] cluster-containing dehydratases was studied. Reactive oxygen species (ROS)-sensitive fumarase A (FumA) and aconitase B (AcnB) as well as ROS-resistant fumarase C (FumC) and aconitase A (AcnA) were assayed in cell-free extracts from tellurite-exposed cells in both the presence and absence of oxygen. While over 90 % of FumA and AcnB activities were lost in the presence of oxygen, no enzyme inactivation was observed in anaerobiosis. This result was not dependent upon protein biosynthesis, as determined using translation-arrested cells. Enzyme activity of purified FumA and AcnB was inhibited when exposed to anin vitrosuperoxide-generating, tellurite-reducing system (ITRS). No inhibitory effect was observed when tellurite was omitted from the ITRS.In vivoandin vitroreconstitution experiments with tellurite-damaged FumA and AcnB suggested that tellurite effects involve [Fe–S] cluster disabling. In fact, after exposing FumA to ITRS, released ferrous ion from the enzyme was demonstrated by spectroscopic analysis using the specific Fe2+chelator 2,2′-bipyridyl. Subsequent spectroscopic paramagnetic resonance analysis of FumA exposed to ITRS showed the characteristic signal of an oxidatively inactivated [3Fe–4S]+cluster. These results suggest that tellurite inactivates enzymes of this kind via a superoxide-dependent disabling of their [4Fe–4S] catalytic clusters.
Functional characterization of FlgM in the regulation of flagellar synthesis and motility in Yersinia pseudotuberculosis
Authors: Ding Lisha , Wang Yao , Hu Yangbo , Atkinson Steve , Williams Paul , Chen Shiyun
Published: June 2009

We describe here the functional characterization of the flgM gene in Yersinia pseudotuberculosis. Direct interaction of FlgM with the alternative sigma factor σ
28 (FliA) was first confirmed. A conserved region in the C-terminus of FlgM was found which included the σ
28 binding domain. By site-directed mutagenesis, bacterial two-hybrid analysis and Western blotting, the primary FlgM binding sites with σ
28 were shown to be Ile85, Ala86 and Leu89. A role for FlgM in swimming motility was demonstrated by inactivation of flgM and subsequent complementation in trans. Transcriptional fusion analyses showed differential gene expression of flhDC, fliA, flgM and fliC in the fliA and flgM mutants compared with the wild-type. flhDC expression was not influenced by σ
28 or FlgM while fliA expression was abolished in the fliA mutant and considerably reduced in the flgM mutant when compared to the wild-type, indicating that both FliA and FlgM can activate fliA transcription. Conversely, flgM transcription was higher in the fliA mutant when compared to the wild-type, suggesting that flgM transcription was repressed by σ
28. Interestingly, fliC expression was markedly increased in the flgM mutant, suggesting a negative regulatory role for FlgM in fliC expression. The transcription of other σ-dependent genes (cheW, flgD, flaA, csrA and fliZ) was also examined in fliA and flgM mutant backgrounds and this revealed that other σ-factors apart from σ
28 may be involved in flagellar biogenesis in Y. pseudotuberculosis. Taking together the motility phenotypes and effects of flgM mutation on the regulation of these key motility genes, we propose that the mechanisms regulating flagellar biogenesis in Y. pseudotuberculosis may differ from those described for other bacteria.
The ecology, epidemiology and virulence of Enterococcus
Authors: Fisher Katie , Phillips Carol
Published: June 2009

Enterococci are Gram-positive, catalase-negative, non-spore-forming, facultative anaerobic bacteria, which usually inhabit the alimentary tract of humans in addition to being isolated from environmental and animal sources. They are able to survive a range of stresses and hostile environments, including those of extreme temperature (5–65 °C), pH (4.5−10.0) and high NaCl concentration, enabling them to colonize a wide range of niches. Virulence factors of enterococci include the extracellular protein Esp and aggregation substances (Agg), both of which aid in colonization of the host. The nosocomial pathogenicity of enterococci has emerged in recent years, as well as increasing resistance to glycopeptide antibiotics. Understanding the ecology, epidemiology and virulence of Enterococcus species is important for limiting urinary tract infections, hepatobiliary sepsis, endocarditis, surgical wound infection, bacteraemia and neonatal sepsis, and also stemming the further development of antibiotic resistance.
Meningococcal biofilm growth on an abiotic surface – a model for epithelial colonization?
Authors: O'Dwyer Clíona A. , Li Ming-Shi , Langford Paul R. , Kroll J. Simon
Published: June 2009

Neisseria meningitidis colonizes the human nasopharynx asymptomatically, often for prolonged periods, but occasionally invades from this site to cause life-threatening infection. In the nasopharynx aggregated organisms are closely attached to the epithelial surface, in a state in which the expression of components of the bacterial envelope differs significantly from that found in organisms multiplying exponentially in liquid phase culture or in the blood. We and others have hypothesized that here they are in the biofilm state, and to explore this we have investigated biofilm formation by the serogroup B strain MC58 on an abiotic surface, in a sorbarod system. Transcriptional changes were analysed, focusing on alteration in gene expression relevant to polysaccharide capsulation, lipooligosaccharide and outer-membrane protein synthesis – all phenotypes of importance in epithelial colonization. We report downregulation of genes controlling capsulation and the production of core oligosaccharide, and upregulation of genes encoding a range of outer-membrane components, reflecting phenotypic changes that have been established to occur in the colonizing state. A limited comparison with organisms recovered from an extended period of co-cultivation with epithelial cells suggests that this model system may better mirror natural colonization than do short-term meningococcal/epithelial cell co-cultivation systems. Modelling prolonged meningococcal colonization with a sorbarod system offers insight into gene expression during this important, but experimentally relatively inaccessible, phase of human infection.
Homocysteine editing and growth inhibition in Escherichia coli
Authors: Sikora Marta , Jakubowski Hieronim
Published: June 2009

InEscherichia colihomocysteine (Hcy) is metabolically converted to the thioester Hcy-thiolactone in ATP-consuming reactions catalysed by methionyl-, isoleucyl- and leucyl-tRNA synthetases. Here we show that growth inhibition caused by supplementation ofE. colicultures with Hcy is accompanied by greatly increased accumulation of Hcy-thiolactone. Energy dissipation for Hcy editing increases 100-fold in the presence of exogenous Hcy and reaches one mole of ATP unproductively dissipated for Hcy-thiolactone synthesis per each mole of ATP that is consumed for methionine activation. Inhibiting Hcy-thiolactone synthesis with isoleucine, leucine or methionine accelerates bacterial growth in Hcy-supplemented cultures. Growth rates in Hcy-inhibited cultures are inversely related to the accumulation of Hcy-thiolactone. We also show that the levels of proteinN-linked Hcy modestly increase inE. colicells in Hcy-supplemented cultures. The results suggest that Hcy editing restrains bacterial growth.
Pseudomonas aeruginosa Las quorum sensing autoinducer suppresses growth and biofilm production in Legionella species
Authors: Kimura Soichiro , Tateda Kazuhiro , Ishii Yoshikazu , Horikawa Manabu , Miyairi Shinichi , Gotoh Naomasa , Ishiguro Masaji , Yamaguchi Keizo
Published: June 2009

Bacteria commonly communicate with each other by a cell-to-cell signalling mechanism known as quorum sensing (QS). Recent studies have shown that the Las QS autoinducer N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) of Pseudomonas aeruginosa performs a variety of functions not only in intraspecies communication, but also in interspecies and interkingdom interactions. In this study, we report the effects of Pseudomonas 3-oxo-C12-HSL on the growth and suppression of virulence factors in other bacterial species that frequently co-exist with Ps. aeruginosa in nature. It was found that 3-oxo-C12-HSL, but not its analogues, suppressed the growth of Legionella pneumophila in a dose-dependent manner. However, 3-oxo-C12-HSL did not exhibit a growth-suppressive effect on Serratia marcescens, Proteus mirabilis, Escherichia coli, Alcaligenes faecalis and Stenotrophomonas maltophilia. A concentration of 50 μM 3-oxo-C12-HSL completely inhibited the growth of L. pneumophila. Additionally, a significant suppression of biofilm formation was demonstrated in L. pneumophila exposed to 3-oxo-C12-HSL. Our results suggest that the Pseudomonas QS autoinducer 3-oxo-C12-HSL exerts both bacteriostatic and virulence factor-suppressive activities on L. pneumophila alone.
Raman spectroscopic typing reveals the presence of carotenoids in Mycoplasma pneumoniae
Authors: Maquelin Kees , Hoogenboezem Theo , Jachtenberg Jan-Willem , Dumke Roger , Jacobs Enno , Puppels Gerwin J. , Hartwig Nico G. , Vink Cornelis
Published: June 2009

Raman spectroscopy has previously been demonstrated to be a highly useful methodology for the identification and/or typing of micro-organisms. In this study, we set out to evaluate whether this technology could also be applied as a tool to discriminate between isolates of Mycoplasma pneumoniae, which is generally considered to be a genetically highly uniform species. In this evaluation, a total of 104 strains of M. pneumoniae were analysed, including two reference strains (strains M129 and FH), and 102 clinical isolates, which were isolated between 1973 and 2005 and originated from various countries. By Raman spectral analysis (Raman typing) of this strain collection, we were able to reproducibly distinguish six different clusters of strains. An unequivocal correlation between Raman typing and P1 genotyping, which is based on sequence differences in the P1 (or MPN141) gene of M. pneumoniae, was not observed. In the two major Raman clusters that we identified (clusters 3 and 6, which together harboured 81 % of the strains), the different P1 subtypes were similarly distributed, and ∼76 % isolates were of subtype 1, ∼20 % of subtype 2 and ∼5 % of variant 2a. Nevertheless, a relatively high prevalence of P1 subtype 2 strains was found in clusters 2 and 5 (100 %), as well as in cluster 1 (75 %) and cluster 4 (71 %); these clusters, however, harboured a small number of strains. Only two of the strains (2 %) could not be typed correctly. Interestingly, analysis of the Raman spectra revealed the presence of carotenoids in M. pneumoniae. This finding is in line with the identification of M. pneumoniae genes that have similarity with genes involved in a biochemical pathway leading to carotenoid synthesis, i.e. the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Therefore, we hypothesize that M. pneumoniae hosts an MEP-like pathway for carotenoid synthesis. We conclude that Raman spectroscopy is a convenient tool for discriminating between M. pneumoniae strains, and that it presents a promising supplement to the current methods for typing of this bacterium.
Transfer, stable maintenance and expression of the mycolactone polyketide megasynthase mls genes in a recombination-impaired Mycobacterium marinum
Authors: Porter Jessica L. , Tobias Nicholas J. , Hong Hui , Tuck Kellie L. , Jenkin Grant A. , Stinear Timothy P.
Published: June 2009

The human pathogenMycobacterium ulceransproduces a polyketide metabolite called mycolactone with potent immunomodulatory activity.M. ulceransstrain Agy99 has a 174 kb plasmid called pMUM001 with three large genes (mlsA1, 51 kb;mlsA2, 7.2 kb;mlsB, 43 kb) that encode type I polyketide synthases (PKS) required for the biosynthesis of mycolactone, as demonstrated by transposon mutagenesis. However, there have been no reports of transfer of themlslocus to another mycobacterium to demonstrate that these genes are sufficient for mycolactone production because in addition to their large size, themlsgenes contain a high level of internal sequence repetition, such that the entire 102 kb locus is composed of only 9.5 kb of unique DNA. The combination of their large size and lack of stability during laboratory passage makes them a challenging prospect for transfer to a more rapidly growing and genetically tractable host. Here we describe the construction of two bacterial artificial chromosomeEscherichia coli/Mycobacteriumshuttle vectors, one based on the pMUM001 origin of replication bearingmlsB, and the other based on the mycobacteriophage L5 integrase, bearingmlsA1andmlsA2. The combination of these two constructs permitted the two-step transfer of the entire 174 kb pMUM001 plasmid toMycobacterium marinum, a rapidly growing non-mycolactone-producing mycobacterium that is a close genetic relative ofM. ulcerans. To improve the stability of themlslocus inM. marinum,recAwas inactivated by insertion of a hygromycin-resistance gene using double-crossover allelic exchange. As expected, the ΔrecAmutant displayed increased susceptibility to UV killing and a decreased frequency of homologous recombination. Southern hybridization and RT-PCR confirmed the stable transfer and expression of themlsgenes in both wild-typeM. marinumand therecAmutant. However, neither mycolactone nor its predicted precursor metabolites were detected in either strain. These experiments show that it is possible to successfully manipulate and stably transfer the largemlsgenes, but that other bacterial host factors appear to be required to facilitate mycolactone production.
Two novel metal-independent long-chain alkyl alcohol dehydrogenases from Geobacillus thermodenitrificans NG80-2
Authors: Liu Xueqian , Dong Yanpeng , Zhang Jing , Zhang Aixiang , Wang Lei , Feng Lu
Published: June 2009

Two alkyl alcohol dehydrogenase (ADH) genes from the long-chain alkane-degrading strainGeobacillus thermodenitrificansNG80-2 were characterizedin vitro. ADH1 and ADH2 were prepared heterologously inEscherichia colias a homooctameric and a homodimeric protein, respectively. Both ADHs can oxidize a broad range of alkyl alcohols up to at least C30, as well as 1,3-propanediol and acetaldehyde. ADH1 also oxidizes glycerol, and ADH2 oxidizes isopropyl alcohol, isoamylol, acetone, octanal and decanal. The best substrate is ethanol for ADH1 and 1-octanol for ADH2. For both ADHs, the optimum assay condition is at 60 °C and pH 8.0, and both NAD and NADP can be used as the cofactor. Sequence analysis reveals that ADH1 and ADH2 belong to the Fe-containing/activated long-chain ADHs. However, the two enzymes contain neither Fe nor other metals, and Fe is not required for the activity, suggesting a new type of ADH. The ADHs characterized here are potentially useful in crude oil bioremediation and other bioconversion processes.
Folding and trimerization of signal sequence-less mature TolC in the cytoplasm of Escherichia coli
Authors: Masi Muriel , Duret Guillaume , Delcour Anne H. , Misra Rajeev
Published: June 2009

TolC is a multifunctional outer-membrane protein (OMP) of Escherichia coli that folds into a unique α/β-barrel structure. Previous studies have shown that unlike the biogenesis of β-barrel OMPs, such as porins, TolC assembles independently from known periplasmic folding factors. Yet, the assembly of TolC, like that of β-barrel OMPs, is dependent on BamA and BamD, two essential components of the β-barrel OMP assembly machinery. We have investigated the folding properties and cellular trafficking of a TolC derivative that lacks the entire signal sequence (TolCΔ2–22). A significant amount of TolCΔ2–22 was found to be soluble in the cytoplasm, and a fraction of it folded and trimerized into a conformation similar to that of the normal outer membrane-localized TolC protein. Some TolCΔ2–22 was found to associate with membranes, but failed to assume a wild-type-like folded conformation. The null phenotype of TolCΔ2–22 was exploited to isolate suppressor mutations, the majority of which mapped in secY. In the secY suppressor background, TolCΔ2–22 resumed normal function and folded like wild-type TolC. Proper membrane insertion could not be achieved upon in vitro incubation of cytoplasmically folded TolCΔ2–22 with purified outer membrane vesicles, showing that even though TolC is intrinsically capable of folding and trimerization, for successful integration into the outer membrane these events need to be tightly coupled to the insertion process, which is mediated by the Bam machinery. Genetic and biochemical data attribute the unique folding and assembly pathways of TolC to its large soluble α-helical domain.
In the non-insect-transmissible line of onion yellows phytoplasma (OY-NIM), the plasmid-encoded transmembrane protein ORF3 lacks the major promoter region
Authors: Ishii Yoshiko , Kakizawa Shigeyuki , Hoshi Ayaka , Maejima Kensaku , Kagiwada Satoshi , Yamaji Yasuyuki , Oshima Kenro , Namba Shigetou
Published: June 2009

‘Candidatus Phytoplasma asteris’, onion yellows strain (OY), a mildly pathogenic line (OY-M), is a phytopathogenic bacterium transmitted by Macrosteles striifrons leafhoppers. OY-M contains two types of plasmids (EcOYM and pOYM), each of which possesses a gene encoding the putative transmembrane protein, ORF3. A non-insect-transmissible line of this phytoplasma (OY-NIM) has the corresponding plasmids (EcOYNIM and pOYNIM), but pOYNIM lacks orf3. Here we show that in OY-M, orf3 is transcribed from two putative promoters and that on EcOYNIM, one of the promoter sequences is mutated and the other deleted. We also show by immunohistochemical analysis that ORF3 is not expressed in OY-NIM-infected plants. Moreover, ORF3 protein seems to be preferentially expressed in OY-M-infected insects rather than in plants. We speculate that ORF3 may play a role in the interactions of OY with its insect host.
Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages
Authors: van der Ploeg Jan R.
Published: June 2009

Clustered regularly interspaced short palindromic repeats (CRISPR) consist of highly conserved direct repeats interspersed with variable spacer sequences. They can protect bacteria against invasion by foreign DNA elements. The genome sequence ofStreptococcus mutansstrain UA159 contains two CRISPR loci, designated CRISPR1 and CRISPR2. The aims of this study were to analyse the organization of CRISPR in furtherS. mutansstrains and to investigate the importance of CRISPR in acquired immunity to M102-like phages. The sequences of CRISPR1 and CRISPR2 arrays were determined for 29S. mutansstrains from different persons. More than half of the CRISPR1 spacers and about 35 % of the CRISPR2 spacers showed sequence similarity with the genome sequence of M102, a virulent siphophage specific forS. mutans. Although only a few spacers matched the phage sequence completely, most of the mismatches had no effect on the amino acid sequences of the phage-encoded proteins. The results suggest thatS. mutansis often attacked by M102-like bacteriophages, and that its acquisition of novel phage-derived CRISPR sequences goes along with the presence ofS. mutansphages in the environment. Analysis of CRISPR1 of M102-resistant mutants ofS. mutansOMZ 381 showed that some of them had acquired novel spacers, and the sequences of all but one of these matched the phage M102 genome sequence. This suggests that the acquisition of the spacers contributed to the resistance against phage infection. However, since not all resistant mutants had new spacers, and since the removal of the CRISPR1 array in one of the mutants and in wild-type strains did not lead to loss of resistance to infection by M102, the acquisition of resistance must be based on further elements as well.
Cloning of feather-degrading minor extracellular protease from Bacillus cereus DCUW: dissection of the structural domains
Authors: Ghosh Abhrajyoti , Chakrabarti Krishanu , Chattopadhyay Dhrubajyoti
Published: June 2009

Bacterial extracellular proteases play an important role in cell survival and cell–cell communication. A high-molecular-mass minor extracellular protease (Vpr) from a feather-degrading bacterium, Bacillus cereus DCUW, has been reported by our laboratory. In the present study, we cloned and expressed Vpr in Escherichia coli. Complete nucleotide sequencing of this gene predicted that the protease is a member of the serine protease family, and smart domain analysis revealed that the protease consists of an N-terminal signal sequence for secretion, a subtilisin_N sequence that is a signature for N-terminal processing, a catalytic S_8 peptidase domain, and finally a long C-terminal protease-associated (PA) region containing nine intrinsically disordered subdomains. Four truncated constructs of the Vpr protease were cloned and expressed in E. coli. We found that the catalytic domain (amino acid residues 172–583) is sufficient for protease activity. Maturation of the Vpr protease needed both N-terminal and C-terminal processing. We have demonstrated that the oligomerization property is associated with the C-terminal protease-associated domain and also shown that the substrate-binding specificity to raw feather resides in this domain.
Contributions of two UDP-glucose dehydrogenases to viability and polymyxin B resistance of Burkholderia cenocepacia
Authors: Loutet Slade A. , Bartholdson S. Josefin , Govan John R. W. , Campopiano Dominic J. , Valvano Miguel A.
Published: June 2009

Burkholderia cenocepaciais highly resistant to antimicrobial peptides and we hypothesized that the conversion of UDP-glucose to UDP-glucuronic acid, a reaction catalysed by the enzyme UDP-glucose dehydrogenase (Ugd) would be important for this resistance. The genome ofB. cenocepaciacontains three predictedugdgenes:ugdBCAL2946,ugdBCAM0855andugdBCAM2034, all of which were individually inactivated. Only inactivation ofugdBCAL2946resulted in increased sensitivity to polymyxin B and this sensitivity could be overcome when eitherugdBCAL2946orugdBCAM0855but notugdBCAM2034was expressed from plasmids. The growth of a conditionalugdBCAL2946mutant, created in the ΔugdBCAM0855background, was significantly impaired under non-permissive conditions. Growth could be rescued by eitherugdBCAL2946orugdBCAM0855expressedin trans, but not byugdBCAM2034. Biochemical analysis of the purified, recombinant forms of UgdBCAL2946and UgdBCAM0855revealed that they are soluble homodimers with similarin vitroUgd activity and comparable kinetic constants for their substrates UDP-glucose and NAD+. Purified UgdBCAM2034showed noin vitroUgd activity. Real-time PCR analysis showed that the expression ofugdBCAL2946was 5.4- and 135-fold greater than that ofugdBCAM0855andugdBCAM2034, respectively. Together, these data indicate that the combined activity of UgdBCAL2946and UgdBCAM0855is essential for the survival ofB. cenocepaciabut only the most highly expressedugdgene,ugdBCAL2946, is required for polymyxin B resistance.
From a consortium sequence to a unified sequence: the Bacillus subtilis 168 reference genome a decade later
Authors: Barbe Valérie , Cruveiller Stéphane , Kunst Frank , Lenoble Patricia , Meurice Guillaume , Sekowska Agnieszka , Vallenet David , Wang Tingzhang , Moszer Ivan , Médigue Claudine , Danchin Antoine
Published: June 2009

Comparative genomics is the cornerstone of identification of gene functions. The immense number of living organisms precludes experimental identification of functions except in a handful of model organisms. The bacterial domain is split into large branches, among which the Firmicutes occupy a considerable space. Bacillus subtilis has been the model of Firmicutes for decades and its genome has been a reference for more than 10 years. Sequencing the genome involved more than 30 laboratories, with different expertises, in a attempt to make the most of the experimental information that could be associated with the sequence. This had the expected drawback that the sequencing expertise was quite varied among the groups involved, especially at a time when sequencing genomes was extremely hard work. The recent development of very efficient, fast and accurate sequencing techniques, in parallel with the development of high-level annotation platforms, motivated the present resequencing work. The updated sequence has been reannotated in agreement with the UniProt protein knowledge base, keeping in perspective the split between the paleome (genes necessary for sustaining and perpetuating life) and the cenome (genes required for occupation of a niche, suggesting here that B. subtilis is an epiphyte). This should permit investigators to make reliable inferences to prepare validation experiments in a variety of domains of bacterial growth and development as well as build up accurate phylogenies.
Identification of novel LPXTG-linked surface proteins from Streptococcus gordonii
Authors: Davies Julia R. , Svensäter Gunnel , Herzberg Mark C.
Published: June 2009

Surface adhesion plays an essential part in the survival of the commensal organism Streptococcus gordonii in the oral cavity as well as during opportunistic infections such as endocarditis. At least two types of cell surface protein involved in adhesion are found on the surface of Gram-positive bacteria: those anchored via an LPXTG motif by the enzyme sortase A (SrtA) and those associated with the cell surface by, as yet, unknown mechanisms. In srtA−
mutants, LPXTG-containing proteins have been shown to be released rather than cross-linked to the cell wall. We have therefore used 2D gel electrophoresis of released proteins from an srtA−
mutant as well as the wild-type strain, followed by peptide identification by MS, to identify a set of novel proteins predicted to be present on the surface of S. gordonii DL1. This includes two large LPXTG-linked proteins (SGO_0707 and SGO_1487), which both contain tandemly repeated sequences similar to those present in known fibrillar adhesins. A 5′-nucleotidase and a protein with a putative collagen-binding domain, both containing LPXTG motifs, were also identified. Anchorless proteins with known chaperone, stress response and elongation factor functions, apparently responsible for bacterial binding to keratinocytes and saliva-coated surfaces in the absence of the LPXTG-linked adhesins, were also associated with the cell surface. These data reveal a range of proteins to be present on the S. gordonii DL1 cell surface, the expression of which plays an important role in adhesion to epithelia and which represent likely candidates for novel virulence factors in S. gordonii.
The OxyR homologue in Tannerella forsythia regulates expression of oxidative stress responses and biofilm formation
Authors: Honma Kiyonobu , Mishima Elina , Inagaki Satoru , Sharma Ashu
Published: June 2009

Tannerella forsythia is an anaerobic periodontal pathogen that encounters constant oxidative stress in the human oral cavity due to exposure to air and reactive oxidative species from coexisting dental plaque bacteria as well as leukocytes. In this study, we sought to characterize a T. forsythia ORF with close similarity to bacterial oxidative stress response sensor protein OxyR. To analyse the role of this OxyR homologue, a gene deletion mutant was constructed and characterized. Aerotolerance, survival after hydrogen peroxide challenge and transcription levels of known bacterial antioxidant genes were then determined. Since an association between oxidative stress and biofilm formation has been observed in bacterial systems, we also investigated the role of the OxyR protein in biofilm development by T. forsythia. Our results showed that aerotolerance, sensitivity to peroxide challenge and the expression of oxidative stress response genes were significantly reduced in the mutant as compared with the wild-type strain. Moreover, the results of biofilm analyses showed that, as compared with the wild-type strain, the oxyR mutant showed significantly less autoaggregation and a reduced ability to form mixed biofilms with Fusobacterium nucleatum. In conclusion, a gene annotated in the T. forsythia genome as an oxyR homologue was characterized. Our studies showed that the oxyR homologue in T. forsythia constitutively activates antioxidant genes involved in resistance to peroxides as well as oxygen stress (aerotolerance). In addition, the oxyR deletion attenuates biofilm formation in T. forsythia.
Effects of altered TatC proteins on protein secretion efficiency via the twin-arginine translocation pathway of Bacillus subtilis
Authors: Eijlander Robyn T. , Kolbusz Magdalena A. , Berendsen Erwin M. , Kuipers Oscar P.
Published: June 2009

Protein translocation via the Tat machinery in thylakoids and bacteria occurs through a cooperation between the TatA, TatB and TatC subunits, of which the TatC protein forms the initial Tat substrate-binding site. The Bacillus subtilis Tat machinery lacks TatB and comprises two separate TatAC complexes with distinct substrate specificities: PhoD is secreted by the TatAdCd complex, whereas YwbN is secreted by the TatAyCy complex. To study the role of the Gram-positive TatC proteins in Tat-dependent protein secretion efficiency, we applied several genetic engineering approaches to modify and analyse the B. subtilis TatCd and TatCy proteins. Cytoplasmic and transmembrane domain exchange between TatCd and TatCy resulted in stable chimeric proteins that were unable to secrete both known substrates of the B. subtilis Tat system. Site-directed mutagenesis of conserved residues in the N-terminal part of both TatC proteins revealed significant differences in the degree of importance of these residues between TatCd, TatCy and Escherichia coli TatC. In addition, two small C-terminal deletions in TatCy completely abolished YwbN translocation, indicating that this terminus is essential for Tat translocation activity. Important differences from previous observations for E. coli TatC and implications for substrate binding and translocation are discussed.
Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly ‘Pseudomonas butanovora’
Authors: Cooley Richard B. , Dubbels Bradley L. , Sayavedra-Soto Luis A. , Bottomley Peter J. , Arp Daniel J.
Published: June 2009

Soluble butane monooxygenase (sBMO), a three-component di-iron monooxygenase complex expressed by the C2–C9alkane-utilizing bacteriumThauera butanivorans, was kinetically characterized by measuring substrate specificities for C1–C5alkanes and product inhibition profiles. sBMO has high sequence homology with soluble methane monooxygenase (sMMO) and shares a similar substrate range, including gaseous and liquid alkanes, aromatics, alkenes and halogenated xenobiotics. Results indicated that butane was the preferred substrate (defined bykcat : Kmratios). Relative rates of oxidation for C1–C5alkanes differed minimally, implying that substrate specificity is heavily influenced by differences in substrateKmvalues. The low micromolarKmfor linear C2–C5alkanes and the millimolarKmfor methane demonstrate that sBMO is two to three orders of magnitude more specific for physiologically relevant substrates ofT. butanivorans. Methanol, the product of methane oxidation and also a substrate itself, was found to have similarKmandkcatvalues to those of methane. This inability to kinetically discriminate between the C1alkane and C1alcohol is observed as a steady-state concentration of methanol during the two-step oxidation of methane to formaldehyde by sBMO. Unlike methanol, alcohols with chain length C2–C5do not compete effectively with their respective alkane substrates. Results from product inhibition experiments suggest that the geometry of the active site is optimized for linear molecules four to five carbons in length and is influenced by the regulatory protein component B (butane monooxygenase regulatory component; BMOB). The data suggest that alkane oxidation by sBMO is highly specialized for the turnover of C3–C5alkanes and the release of their respective alcohol products. Additionally, sBMO is particularly efficient at preventing methane oxidation during growth on linear alkanes ≥C2,despite its high sequence homology with sMMO. These results represent, to the best of our knowledge, the first kineticin vitrocharacterization of the closest known homologue of sMMO.
Transmembrane topology of the AbsA1 sensor kinase of Streptomyces coelicolor
Authors: McKenzie Nancy L. , Nodwell Justin R.
Published: June 2009

The sensor kinase AbsA1 (SCO3225) phosphorylates the response regulator AbsA2 (SCO3226) and dephosphorylates AbsA2∼P. The phosphorylated response regulator represses antibiotic biosynthesis operons in Streptomyces coelicolor. AbsA1 was predicted to have an atypical transmembrane topology, and the location of its signal-sensing domain is not readily obvious. To better understand this protein and to gain insight into its signal response mechanism, we determined its transmembrane topology using fusions of absA1 to egfp, which is believed to be the first application of this approach to transmembrane topology in the actinomycetes. Our results are in agreement with the in silico topological predictions and demonstrate that AbsA1 has five transmembrane domains, four near the N terminus and one near the C terminus. Unlike most sensor kinases, the largest extracellular portion of AbsA1 is at the C terminus.
polR, a pathway-specific transcriptional regulatory gene, positively controls polyoxin biosynthesis in Streptomyces cacaoi subsp. asoensis
Authors: Li Rui , Xie Zhoujie , Tian Yuqing , Yang Haihua , Chen Wenqing , You Delin , Liu Gang , Deng Zixin , Tan Huarong
Published: June 2009

The polyoxin (POL) biosynthetic gene cluster (pol) was recently cloned from Streptomyces cacaoi subsp. asoensis. A 3.3 kb DNA fragment carrying an obvious open reading frame (polR), whose deduced product shows sequence similarity to SanG of Streptomyces ansochromogenes and PimR of Streptomyces natalensis, was revealed within the pol gene cluster. Disruption of polR abolished POL production, which could be complemented by the integration of a single copy of polR into the chromosome of the non-producing mutant. The introduction of an extra copy of polR in the wild-type strain resulted in increased production of POLs. The transcription start point (tsp) of polR was determined by S1 mapping. Reverse transcriptase PCR experiments showed that PolR is required for the transcription of 18 structural genes in the pol gene cluster. Furthermore, we showed that polC and polB, the respective first genes of two putative operons (polC–polQ2 and polA–polB) consisting of 16 and 2 of these 18 genes, have similar promoter structures. Gel retardation assays indicated that PolR has specific DNA-binding activity for the promoter regions of polC and polB. Our data suggest that PolR acts in a positive manner to regulate POL production by activating the transcription of at least two putative operons in the pol gene cluster.
The GPI-modified proteins Pga59 and Pga62 of Candida albicans are required for cell wall integrity
Authors: Moreno-Ruiz Emilia , Ortu Giuseppe , de Groot Piet W. J. , Cottier Fabien , Loussert Céline , Prévost Marie-Christine , de Koster Chris , Klis Frans M. , Goyard Sophie , d'Enfert Christophe
Published: June 2009

The fungal cell wall is essential in maintaining cellular integrity and plays key roles in the interplay between fungal pathogens and their hosts. ThePGA59andPGA62genes encode two short and related glycosylphosphatidylinositol-anchored cell wall proteins and their expression has been previously shown to be strongly upregulated when the human pathogenCandida albicansgrows as biofilms. Using GFP fusion proteins, we have shown that Pga59 and Pga62 are cell-wall-located,N- andO-glycosylated proteins. The characterization ofC. albicans pga59Δ/pga59Δ,pga62Δ/pga62Δ andpga59Δ/pga59Δpga62Δ/pga62Δ mutants suggested a minor role of these two proteins in hyphal morphogenesis and that they are not critical to biofilm formation. Importantly, the sensitivity to different cell-wall-perturbing agents was altered in these mutants. In particular, simultaneous inactivation ofPGA59andPGA62resulted in high sensitivity to Calcofluor white, Congo red and nikkomicin Z and in resistance to caspofungin. Furthermore, cell wall composition and observation by transmission electron microscopy indicated an altered cell wall structure in the mutant strains. Collectively, these data suggest that the cell wall proteins Pga59 and Pga62 contribute to cell wall stability and structure.
Prenatal Intervention for Lower Urinary Tract Obstruction
Authors: Kitchens David M. , Herndon C. D. Anthony
Published: June 2009

Hydronephrosis is one of the most common abnormalities detected on routine prenatal ultrasounds, being noted in up to 1% of fetuses. Rarely, severe hydronephrosis coexists with oligohydramnios, which portends a poor prognosis. We review the most recent literature on the results of prenatal intervention in this setting. Presently, the first randomized controlled trial to address whether prenatal vesicoamniotic shunting improves survival and renal function is underway, and should address the value of prenatal intervention.
Postnatal Imaging of Antenatal Hydronephrosis
Authors: Kitchens David M. , Herndon C. D. Anthony
Published: June 2009

Radiologic imaging of the newborn detected prenatally with hydronephrosis should follow a systematic approach. Upper and lower urinary tract imaging should be performed in most cases in order to determine the etiology and gauge the use of future imaging. An overview of renal ultrasound, voiding cystourethrography, renal scintigraphy, and magnetic resonance urography in the setting of antenatal hydronephrosis are discussed.
The Management of Ureteropelvic Junction Obstruction Presenting with Prenatal Hydronephrosis
Authors: Herndon C.D. Anthony , Kitchens David M.
Published: June 2009

The treatment of the newborn diagnosed with a ureteropelvic obstruction prenatally should follow a systematic approach. Although a majority of patients can be followed without surgical intervention, controversy exists concerning appropriate follow-up. Furthermore, a significant number of patients will manifest mild disease and thus deserve abbreviated follow-up. Herein, an appropriate algorithm and a review of the literature are discussed.
Biophysical and Structural Studies on the Capsid Protein of the Human Immunodeficiency Virus Type 1: A New Drug Target?
Authors: Neira José L.
Published: June 2009

AIDS affects 30 million people worldwide and is one of the deadliest epidemics in human history. It is caused by a retrovirus, HIV, whose mature capsid (enclosing the RNA with other proteins) is formed by the assembly of several hundred copies of a protein, CA*. The C-terminal domain of such protein, CAC, is a driving force in virus assembly and the connections in the mature capsid lattice indicate that CAC joins through homodimerization of the CA hexamers. In the first part of this work, I shall review the biophysical studies carried out with the dimeric wild-type CAC protein and a mutant monomeric variant. The results open new venues for the development of drugs able to interact either with the dimeric species, hampering its assembly, or with the monomeric species, obstructing its folding. In the second part of this review, I shall describe the structures of complexes of CAC with small molecules able to weaken its dimerization. Furthermore, interactions with other proteins and lipids are also described. The whole set of results suggests that much of the surface of CAC does not accommodate binding per se, but rather binding sites in the protein are predefined, i.e., there are “hot” spots for binding in CAC (whatever be the molecule to bind). These “hot” residues involve most of the dimerization interface (an α-helix) of the CAC wild-type protein, but also polypeptide patches at the other helices.
Minimum Information About a Microarray Experiment (MIAME) – Successes, Failures, Challenges
Authors: Brazma Alvis
Published: June 2009

The Minimum Information About a Microarray Experiment (known as MIAME) guidelines describe information that needs to be provided to enable the interpretation of the results of a microarray-based experiment unambiguously. The MIAME guidelines were developed by the Microarray Gene Expression Data (MGED) Society. Since the MIAME position paper was published in 2001, it has been cited in the scientific literature well over a thousand times. MIAME has been replicated for many other technologies, the major data repositories are supporting MIAME, and most scientific journals have adopted MIAME guidelines as a requirement for publishing. With the advent of new-generation sequencing technology, MIAME faces new challenges. To address this, the MGED Society has proposed new guidelines, i.e., Minimum Information about a high-throughput SeQuencing Experiment (MINSEQE). Here we present analysis of the reasons for the success of MIAME, as well as discuss where it has failed, and the challenges it faces.