Pirellulosomes: A new type of membrane-bounded cell compartment in planctomycete bacteria of the genus Pirellula

A distinct type of cellular organization was found in two species of the planctomycete genus Pirellula, Pirellula marina and Pirellula staleyi. Both species possess two distinct regions within the cell which are separated by a single membrane. The major region of the cell, the pirellulosome, contains the fibrillar condensed nucleoid. The other area, the polar cap region, forms a continuous layer surrounding the entire pirellulosome and displays a cap of asymmetrically distributed material at one cell pole. Immuno- and cytochemical-labelling of P. marina demonstrated that DNA is located exclusively within the pirellulosome; cell RNA is concentrated in the pirellulosome, with some RNA also located in the polar cap region.

Transfer of Blastobacter natatorius (Sly 1985) to the genus Blastomonas gen nov as Blastomonas natatoria comb nov

The budding bacterium Blastobacter natatorius belongs to the alpha-4 group of the Proteobacteria and clusters phylogenetically on a deep branch with Sphingomonas capsulata, with which it shares 93.9% 16S rRNA sequence similarity. On phylogenetic, phenotypic, and chemotaxonomic grounds a proposal is made to transfer B. natatorius to the genus Blastomonas gen, nov. as Blastomonas natatoria comb, nov.

Cross-linking studies and membrane localization and assembly of radiolabelled large mechanosensitive ion channel (MscL) of Escherichia coli

The gene encoding the large conductance mechanosensitive ion channel (MscL) of Escherichia coli and several deletion mutants of mscL were cloned under the control of the T7 RNA polymerase promoter. Transformation of these constructs into an E. coli strain carrying an inducible T7 RNA polymerase gene allowed the specific production and labelling of MscL with [S-35]methionine. Preparation of membrane fractions of E. coli cells by sucrose gradient centrifugation indicated that the radiolabelled MscL was present in the inner cytoplasmic membrane in agreement with results of several studies. However, treatment of the labelled cells and cell membrane vesicles with various cross-linkers resulted in the majority of labelled protein migrating as a monomer with a small proportion of molecules (approximate to 25%) migrating as dimers and higher order multimers. This result is in contrast with a finding of a study suggesting that the channel exclusively forms hexamers in the cell membrane off. coli (1) and therefore may have profound implication for the activation and/or ''multimerization'' of the channel by mechanical stress exerted to the membrane. In addition, from the specific activity of the radiolabelled protein and the amount of protein in the cytoplasmic membrane fraction we estimated the number of MscL ion channels expressed under these conditions to be approximately 50 channels per single bacterium. (C) 1997 Academic Press.

Mechanisms of biocontrol by Pantoea dispersa of sugar cane leaf scald disease caused by Xanthomonas albilineans

Albicidins, a family of phytotoxins and antibiotics produced by Xanthomonas albilineans, are important in sugar cane leaf scald disease development. The albicidin detoxifying bacterium Pantoea dispersa (syn. Erwinia herbicola) SB1403 provides very effective biocontrol against leaf scald disease in highly susceptible sugar cane cultivars. The P. dispersa gene (albD) for enzymatic detoxification of albicidin has recently been cloned and sequenced. To test the role of albicidin detoxification in biocontrol of leaf scald disease, albD was inactivated in P. dispersa by site-directed mutagenesis. The mutants, which were unable to detoxify albicidin, were less resistant to the toxin and less effective in biocontrol of leaf scald disease than their parent strain. This indicates that albicidin detoxification contributes to the biocontrol capacity of P. dispersa against X. albilineans. Rapid growth and ability to acidify media are other characteristics likely to contribute to the competitiveness of P. dispersa against X. albilineans at wound sites used to invade sugar cane.

Structure of TcpG, the DsbA protein folding catalyst from Vibrio cholerae

The efficient and correct folding of bacterial disulfide bonded proteins in vivo is dependent upon a class of periplasmic oxidoreductase proteins called DsbA, after the Escherichia coli enzyme. In the pathogenic bacterium Vibrio cholerae, the DsbA homolog (TcpG) is responsible for the folding, maturation and secretion of virulence factors. Mutants in which the tcpg gene has been inactivated are avirulent; they no longer produce functional colonisation pill and they no longer secrete cholera toxin. TcpG is thus a suitable target for inhibitors that could counteract the virulence of this organism, thereby preventing the symptoms of cholera. The crystal structure of oxidized TcpG (refined at a resolution of 2.1 Angstrom) serves as a starting point for the rational design of such inhibitors. As expected, TcpG has the same fold as E. coli DsbA, with which it shares similar to 40% sequence identity. Ln addition, the characteristic surface features of DsbA are present in TcpG, supporting the notion that these features play a functional role. While the overall architecture of TcpG and DsbA is similar and the surface features are retained in TcpG, there are significant differences. For example, the kinked active site helix results from a three-residue loop in DsbA, but is caused by a proline in TcpG (making TcpG more similar to thioredoxin in this respect). Furthermore, the proposed peptide binding groove of TcpG is substantially shortened compared with that of DsbA due to a six-residue deletion. Also, the hydrophobic pocket of TcpG is more shallow and the acidic patch is much less extensive than that of E. coli DsbA. The identification of the structural and surface features that are retained or are divergent in TcpG provides a useful assessment of their functional importance in these protein folding catalysts and is an important prerequisite for the design of TcpG inhibitors. (C) 1997 Academic Press Limited.

A novel method for development of species and strain-specific DNA probes and PCR primers for identifying Burkholderia solanacearum (formerly Pseudomonas solanacearum)

Six Burkholderia solanacearum (formerly Pseudomonas solanacearum) genomic DNA fragments were isolated, using RAPD techniques and cloning, from the three genetically diverse strains: ACH092 (Biovar 4), ACH0158 (Biovar 2) and ACH0171 (Biovar 3) (1). One of these cloned fragments was selected because it was present constantly in all bacterial strains analysed. The remaining five clones were selected because Southern hybridisation revealed that each showed partial or complete specificity towards the strain of origin. A seventh genomic fragment showing a strain-specific distribution in Southern hybridisations was obtained by differential restriction, hybridisation and cloning of genomic DNA. Each of these clones was sequenced and primers to amplify the insert were designed. When DNA from the strain of origin was used as template, PCR amplification for each of these fragments yielded a single band on gel analysis. One pair of primers amplified the species-constant fragment of 281 bp from DNA of all B. solanacearum strains investigated, from DNA of the closely related bacterium which causes ''blood disease'' of banana (BDB) and in P. syzigii. The sensitivity of detection of B. solanacearum using these ubiquitous primers was between 1.3 and 20 bacterial cells. The feasibility and reliability of a PCR approach to detection and identification of B. solanacearum was tested in diverse strains of the bacterium in several countries and laboratories.

Lack of galectin-3 alters the balance of innate immune cytokines and confers resistance to Rhodococcus equi infection

Galectin-3 is a p-galactoside-binding lectin implicated in the fine-tuning of innate immunity. Rhodococcus equi, a facultative intracellular bacterium of macrophages, causes severe granulomatous bronchopneumonia in young horses and immunocompromised humans. The aim of this study is to investigate the role of galectin-3 in the innate resistance mechanism against R. equi infection. The bacterial challenge of galectin-3-deficient mice (gal3(-/-)) and their wild-type counterpart (gal3(+/+)) revealed that the LD50 for the gal3(-/-) mice was about seven times higher than that for the gal3(+/+) mice. When challenged with a sublethal dose, gal3(-/-) mice showed lower bacteria counts and higher production of IL-12 and IFN-gamma production, besides exhibiting a delayed although increased inflammatory reaction. Gal3(-/-) macrophages exhibited a decreased frequency of bacterial replication and survival, and higher transcript levels of IL-1 beta, IL-6, IL-10, TLR2 and MyD88. R. equi-infected gal3(+/+) macrophages showed decreased expression of TLR2, whereas R. equi-infected gal3(-/-) macrophages showed enhanced expression of this receptor. Furthermore, galectin-3 deficiency in macrophages may be responsible for the higher IL-1 beta serum levels detected in infected gal3(-/-) mice. Therefore galectin-3 may exert a regulatory role in innate immunity by diminishing IL-1 beta production and thus affecting resistance to R. equi infection.

Oral cavity is not a reservoir for Helicobacter pylori in infected patients with functional dyspepsia

Helicobacter pylori infection is very prevalent in Brazil, infecting almost 65% of the population. The aim of this study was to evaluate the presence of this bacterium in the oral cavity of patients with functional dyspepsia (epigastric pain syndrome), establish the main sites of infection in the mouth, and assess the frequency of cagA and vacA genotypes of oral H. pylori. All 43 outpatients with epigastric pain syndrome, who entered the study, were submitted to upper gastrointestinal endoscopy to rule out organic diseases. Helicobacter pylori infection in the stomach was confirmed by a rapid urease test and urea breath tests. Samples of saliva, the tongue dorsum and supragingival dental plaque were collected from the oral cavity of each subject and subgingival dental plaque samples were collected from the patients with periodontitis; H. pylori infection was verified by polymerase chain reaction using primers that amplify the DNA sequence of a species-specific antigen present in all H. pylori strains; primers that amplify a region of urease gene, and primers for cagA and vacA (m1, m2, s1a, s1b, s2) genotyping. Thirty patients harbored H. pylori in the stomach, but it was not possible to detect H. pylori in any oral samples using P1/P2 and Urease A/B. The genotype cagA was also negative in all samples and vacA genotype could not be characterized (s-m-). The oral cavity may not be a reservoir for H. pylori in patients with epigastric pain syndrome, the bacterium being detected exclusively in the stomach.

Intestinal mucosa-associated microflora in ulcerative colitis patients before and after restorative proctocolectomy with an ileoanal pouch

PURPOSE: This study was designed to identify the mucosa-associated microflora in patients with severe ulcerative colitis before and after restorative proctocolectomy with ileoanal pouch construction in comparison with historic controls. METHODS: Ten patients with a diagnosis of ulcerative colitis were evaluated. Mucus was collected during colonoscopy from all segments of the colon and terminal ileum before surgery, and from the ileal pouch two and eight months after ileostomy closure. The prevalence and mean concentration of the mucosa-associated microflora were compared over time and with historic controls. RESULTS: Veillonella sp was the most prevalent bacterium in patients and controls. Klebsiella sp was significantly more prevalent in the ileum of controls, was not found in patients with ulcerative colitis, and after proctocolectomy returned to values found in controls. Some bacteria such as Enterobacter sp, Staphylococcus sp (coag-), Bacteroides sp (npg), Lactobacillus sp, and Veillonella sp had higher mean concentrations in the ileal pouch of patients after surgery than in controls. CONCLUSION: No bacterium was identified that could be exclusively responsible for the maintenance of the inflammatory process. The mucosa-associated microflora of patients with ulcerative colitis underwent significant changes after proctocolectomy with ileal pouch construction and returned to almost normal values for some bacteria.

The ceja-1 sequence as a potential new molecular marker for Paracoccidioides brasiliensis infection

P>We have developed a two-step PCR assay that amplifies a region of the ceja-1 sequence that is specific for virulent strains of Paracoccidioides brasiliensis. An internal region of the ceja-1 sequence was chosen for designing primers that were utilised in a single tube heminested PCR protocol to amplify DNA from six virulent strains. PCR specificity was determined by the absence of amplified products with genomic DNA from four non-virulent strains of P. brasiliensis and from eight fungal pathogens, one bacterium, two protozoa, one worm and mouse and human genomic DNA (leucocytes). The fact that the PCR product was only obtained with the genetic material from virulent isolates of P. brasiliensis suggested that this partial amplified sequence might be a marker of virulence for this fungus. The diagnostic potential of this PCR was confirmed by the successful amplification of this fragment with genomic DNA obtained in lymph node aspirate from a patient with paracoccidioidomycosis.

The pattern recognition receptors Nod1 and Nod2 account for neutrophil recruitment to the lungs of mice infected with Legionella pneumophila

The intracellular bacterium Legionella pneumophila induces a severe form of pneumonia called Legionnaires diseases, which is characterized by a strong neutrophil (NE) infiltrate to the lungs of infected individuals. Although the participation of pattern recognition receptors, such as Toll-like receptors, was recently demonstrated, there is no information on the role of nod-like receptors (NLRs) for bacterial recognition in vivo and for NE recruitment to the lungs. Here, we employed a murine model of Legionnaires disease to evaluate host and bacterial factors involved in NE recruitment to the mice lungs. We found that L. pneumophila type four secretion system, known as Dot/Icm, was required for NE recruitment as dot/icm mutants fail to trigger NE recruitment in a process independent of bacterial multiplication. By using mice deficient for Nod1, Nod2, and Rip2, we found that these receptors accounted for NE recruitment to the lungs of infected mice. In addition, Rip2-dependent responses were important for cytokine production and bacterial clearance. Collectively, these studies show that Nod1, Nod2, and Rip2 account for generation of innate immune responses in vivo, which are important for NE recruitment and bacterial clearance in a murine model of Legionnaires diseases. (C) 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Septic shock caused by Plesiomonas shigelloides in a patient with sickle beta-zero thalassemia

Invasive infection and extraintestinal complications are rarely caused by Plesiomonas shigelloides, a water-borne bacterium belonging to the Vibrionaceae family. We report a case of a 16-year-old female patient with sickle beta-zero thalassemia who survived septic shock caused by P. shigelloides associated with secondary acute respiratory distress syndrome and disseminated intravascular coagulation. Treatment with a carbapenem was successful, and the patient recovered without any sequelae. The previous reports of P. shigelloides sepsis are cited, and possible pathogenic mechanisms are discussed. (Heart Lung (R) 2010;39:335-339.)

Phenotypic and molecular characterization of recent and archived Erysipelothrix spp. isolated from Brazilian swine

One hundred fifty-one Erysipelothrix spp. isolates from Brazilian swine were characterized by serotyping, determination of antimicrobial susceptibility, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE). Among all isolates, 139 were classified in 18 different serotypes and serotype 2b was the most frequent. The susceptibility profiles of the isolates were very similar among each other, which did not permit subtyping Erysipelothrix spp. isolates by the antimicrobial susceptibility testing. Despite the fact that AFLP and PFGE provided the same discriminatory index (0.98), PFGE was more discriminatory than AFLP, given the types of groups it generates. Regardless the technique employed (AFLP or PFGE), no discrimination between recent and historical isolates was established, neither a fixed epidemiologic pattern for their grouping was observed. Nevertheless, AFLP could be an interesting alternative for discriminating the Erysipelothrix species, while PFGE could be an indication for discerning this bacterium according to the serotypes. (C) 2011 Elsevier Inc. All rights reserved.

Peripheral nervous system and grounds for the neural insult in leprosy

The mechanism of interaction between Mycobacterium leprae and neural cells has not been elucidated so far. No satisfactory interpretation exists as to the bacterium tropism to the peripheral nervous system in particular. The present study is a review of the micro-physiology of the extracellular apparatus attached to Schwann cells, as well as on the description of morphological units probably involved in the process of the binding to the bacterial wall.

The essential role of toll like receptor-4 in the control of Aggregatibacter actinomycetemcomitans infection in mice

Objective: Aggregatibacter actinomycetemcomitans is an oral Gram-negative bacterium that contributes to periodontitis progression. Isolated antigens from A. actinomycetemcomitans could be activating innate immune cells through Toll-like receptors (TLRs). In this study, we evaluated the role of TLR4 in the control of A. actinomycetemcomitans infection. Material and Methods: We examined the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR4(-/-) mice. The production of cytokines was evaluated by ELISA. The bacterial load was determined by counting the number of colony-forming units per gram of tissue. Results: The results showed that TLR4-deficient mice developed less severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly lower bone loss and inflammatory cell migration to periodontal tissues. However, the absence of TLR4 facilitated the A. actinomycetemcomitans dissemination. Myeloperoxidase activity was diminished in the periodontal tissue of TLR4(-/-) mice. We observed a significant reduction in the production of tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1 beta in the periodontal tissue of TLR4(-/-) mice. Conclusion: The results of this study highlighted the role of TLR4 in controlling A. actinomycetemcomitans infection.