Influence of polysorbate 80 and cyclopropane fatty acid synthase activity on lactic acid production by Lactobacillus casei ATCC 334 at low pH.

Lactic acid is an important industrial chemical commonly produced through microbial fermentation. The efficiency of acid extraction is increased at or below the acid's pKa (pH 3.86), so there is interest in factors that allow for a reduced fermentation pH. We explored the role of cyclopropane synthase (Cfa) and polysorbate (Tween) 80 on acid production and membrane lipid composition in Lactobacillus casei ATCC 334 at low pH. Cells from wild-type and an ATCC 334 cfa knockout mutant were incubated in APT broth medium containing 3 % glucose plus 0.02 or 0.2 % Tween 80. The cultures were allowed to acidify the medium until it reached a target pH (4.5, 4.0, or 3.8), and then the pH was maintained by automatic addition of NH4OH. Cells were collected at the midpoint of the fermentation for membrane lipid analysis, and media samples were analyzed for lactic and acetic acids when acid production had ceased. There were no significant differences in the quantity of lactic acid produced at different pH values by wild-type or mutant cells grown in APT, but the rate of acid production was reduced as pH declined. APT supplementation with 0.2 % Tween 80 significantly increased the amount of lactic acid produced by wild-type cells at pH 3.8, and the rate of acid production was modestly improved. This effect was not observed with the cfa mutant, which indicated Cfa activity and Tween 80 supplementation were each involved in the significant increase in lactic acid yield observed with wild-type L. casei at pH 3.8.

Genetic diversity in proteolytic enzymes and amino acid metabolism among Lactobacillus helveticus strains.

Lactobacillus helveticus CNRZ 32 is recognized for its ability to decrease bitterness and accelerate flavor development in cheese, and has also been shown to release bioactive peptides in milk. Similar capabilities have been documented in other strains of Lb. helveticus, but the ability of different strains to affect these characteristics can vary widely. Because these attributes are associated with enzymes involved in proteolysis or AA catabolism, we performed comparative genome hybridizations to a CNRZ 32 microarray to explore the distribution of genes encoding such enzymes across a bank of 38 Lb. helveticus strains, including 2 archival samples of CNRZ 32. Genes for peptidases and AA metabolism were highly conserved across the species, whereas those for cell envelope-associated proteinases varied widely. Some of the genetic differences that were detected may help explain the variability that has been noted among Lb. helveticus strains in regard to their functionality in cheese and fermented milk.

Diversity of soil mycobacterium isolates from three sites that degrade polycyclic aromatic hydrocarbons.

AIMS: This paper investigates the diversity of polycyclic aromatic hydrocarbon (PAH)-degrading mycobacterium isolates from three different sites within United States: Montana, Texas and Indiana. METHODS AND RESULTS: All five mycobacterium isolates differed in chromosomal restriction enzyme-fragmentation patterns; three isolates possessed linear plasmids. The DNA sequence between the murA and rRNA genes were divergent but the sequence upstream of nidBA genes, encoding a dioxygenase involved in pyrene oxidation, was more highly conserved. Long-chain fatty acid analysis showed most similarity between three isolates from the same Montana site. All isolates were sensitive to rifampicin and isoniazid, used in tuberculosis treatment, and to syringopeptins, produced by plant-associated pseudomonads. Biofilm growth was least for isolate MCS that grew on plate medium as rough-edged colonies. The patterns of substrate utilization in Biolog plates showed clustering of the Montana isolates compared with Mycobacterium vanbaalenii and Mycobacterium gilvum. CONCLUSION: The five PAH-degrading mycobacterium isolates studied differ in genetic and biochemical properties. SIGNIFICANCE AND IMPACT OF THE STUDY: Different properties with respect to antibiotic susceptibility, substrate utilization and biofilm formation could influence the survival in soil of the microbe and their suitability for use in bioaugmentation.

Acute ventricular hemorrhage in adults with hydrocephalus managed by corpus callosotomy and fenestration of the septum pellucidum. Report of three cases.

Three patients with hypertension-induced basal ganglia or thalamic hemorrhage and ventricular rupture underwent corpus callosotomy and fenestration of the septum pellucidum. A patient with a left thalamic hemorrhage underwent surgery on an emergency basis and made a complete physical recovery, although she retained mild psychomotor deficits. Another patient with a large right basal ganglia hemorrhage who also underwent surgery on an emergency basis retained a spastic left hemiparesis without evident psychomotor deficits. The third patient with a left thalamic and basal ganglia hemorrhage, who was initially awake and then lapsed into stupor days later, underwent surgery, but did not recover consciousness. Hydrocephalus was reversed and effectively controlled in all three patients without having to perform a shunt placement procedure.

A concise guide to cDNA microarray analysis.

Microarray expression analysis has become one of the most widely used functional genomics tools. Efficient application of this technique requires the development of robust and reproducible protocols. We have optimized all aspects of the process, including PCR amplification of target cDNA clones, microarray printing, probe labeling and hybridization, and have developed strategies for data normalization and analysis.

Assessing the in vitro cell based ocular compatibility of contact lens materials.

A series of in vitro assays for determining the biocompatibility of ocular biomaterials have been developed and used to assess the differences in performance of omafilcon A, etafilcon A and nelfilcon A contact lens materials. The assays assessed bacterial attachment, macrophage adhesion, granulocyte adhesion and activation, epithelial cell adhesion and corneal cell contact damage. Overall, omafilcon A was found to be more biocompatible than the other materials although there was no significant difference between the epithelial cell adhesion and granulocyte adhesion and activation on any of the hydrogels. Etailcon A performed less well compared to nelfilcon A and omafilcon A with respect macrophage adhesion and bacterial adhesion. The results indicate that these biological assays can be successfully applied for the testing of contact lens materials and may be particularly useful in the in vitro screening of new extended wear contact lens materials where cell adhesion and activation may have a greater influence on clinical performance.

Dictyostelium discoideum nuclear plasmid Ddp5 is a chimera related to the Ddp1 and Ddp2 plasmid families.

The 14,955-bp Dictyostelium discoideum nuclear plasmid Ddp5 contains six transcribed open reading frames. One of these is related to the rep gene of the Ddp2 plasmid, and the other five are related to genes present on the Ddp1 plasmid. The absence of a homolog of the Ddp1 G1 gene, coupled with the presence of the Ddp2 rep gene homolog and of a 1.6-kb inverted repeat analogous to the inverted repeats on members of the Ddp2 plasmid family, suggests that Ddp5 uses Ddp2-like replication and copy number control mechanisms and that it should be assigned to the Ddp2 plasmid family. Ddp5 carries genes homologous to the D1/D3 and D2 genes of the Ddp1 plasmid as well as the Ddp1 G2/G3/D4, G5/D6, and G6/G4/D5 genes. The products of the Ddp5 G2-like, G5-like, and G6-like genes are likely to be transcription factors regulating the expression of themselves and of the other Ddp5 genes. The D1-like and D2-like genes may confer a selective advantage to plasmid-bearing cells, because they can be deleted from plasmid-based shuttle vectors with no apparent effect on vector maintenance. Updated sequence information for the Ddp1 G5/D6, D1/D3, and D2 genes as well as the Dmp1 and Dmp2 G5-like genes is presented. The locations of introns in the G5-like and D1-like genes of Ddp5 and in the homologous genes of the Ddp1, Dmp1, and Dmp2 plasmids were identified. These introns all have GU at the 5' intron border and AG at the 3' intron border, are short (59 to 71 nucleotides), and are AT-rich. A conserved HHCC domain was identified in the G5 proteins; this is a putative zinc binding domain and may be involved in protein-DNA interaction.

Priming of neutrophil respiratory burst activity by lipopolysaccharide from Burkholderia cepacia.

Neutrophil activation may play an important role in the pathogenesis of respiratory disease in Burkholderia cepacia-colonized cystic fibrosis (CF) patients. As bacterial lipopolysaccharides (LPS) are potent immunostimulatory molecules, we investigated the role of B. cepacia LPS in neutrophil activation processes. LPS extracted from a highly transmissible and virulent strain of B. cepacia (J2315) was found to increase neutrophil surface expression of the beta2 integrin, complement receptor 3, and to prime neutrophil respiratory burst responses to the neutrophil-activating agent fMet-Leu-Phe. By contrast, LPS extracted from a nonmucoid Pseudomonas aeruginosa strain isolated from a patient with CF showed little or no priming activity. As B. cepacia is currently being developed as a biocontrol agent for large-scale agricultural release, we compared LPS molecules from a range of bacterial strains for their proinflammatory ability. Priming activity was demonstrated in LPS extracts from all B. cepacia strains tested, with one environmental strain, J2552, showing the highest activity. These findings indicate (i) that B. cepacia LPS may contribute to the inflammatory nature of B. cepacia infection in CF patients, both by promoting increased neutrophil recruitment and by priming neutrophil respiratory burst responses, and (ii) that environmental strains of B. cepacia may have considerable inflammatory potential in susceptible individuals.

Characterisation of a lipoprotein in Mycobacterium bovis (BCG) with sequence similarity to the secreted protein MPB70.

The monoclonal antibody, mAb3C4, raised against sonicated Mycobacterium bovis (Mb) BCG (Tokyo strain 172) cells recognises a 23-kDa protein in the cell wall. The gene encoding this protein was cloned and sequenced and found to be 100% homologous to mpb83 and mpt83 and the putative protein to have a 76% sequence similarity to the secreted, Mb-specific protein, MPB70. MPB83 contains the amino acid (aa) sequence LAGC, which corresponds to the consensus sequence for bacterial lipoprotein modification and processing. MPB83 associated with the detergent phase when separated with Triton X-114 confirming that it is a lipoprotein. When the putative site of acylation, the Cys in the sequence LAGC, was substituted with Ser, the mutated MPB83 associated with the aqueous phase. The cloned gene was used to determine the distribution of mpb83 in various Mycobacterium species. The gene was present in the M. tuberculosis (Mt) complex organisms, as well as in M. kansasii. In addition, Southern blot analysis of Mb and Mt DNA indicated that the mpb83 and mpb70 genes are located close to each other on the genome. Western blot analysis of cell lysates of various Mycobacterium species indicated that only Mt H37Rv and H37Ra produced proteins which reacted with mAb3C4. Furthermore, only two out of six of the Mb field isolates produced detectable antigen, indicating that expression of the mpb83 gene is variable within the Mt complex organisms.

An outbreak of penicillin resistant Streptococcus pneumoniae investigated by a polymerase chain reaction based genotyping method.

AIMS: To characterise the genotypes of penicillin resistant Streptococcus pneumoniae infecting patients in a care of the elderly ward and to study its transmission in a hospital environment. METHODS: Isolates of S pneumoniae were cultured from specimens obtained from patients who had been admitted to a care of the elderly ward where an outbreak had occurred. Penicillin resistant S pneumoniae were also obtained from a series of surveillance throat swabs taken from patients in the same ward. In addition, all penicillin resistant S pneumoniae isolated from specimens submitted for culture at the time of the outbreak were included. Four sensitive strains isolated from a routine microbiology laboratory were included as controls. A simple polymerase chain reaction (PCR) based genotyping method for the penicillin binding protein (PBP) genes 1a, 2x, and 2b was used to characterise the genotypes. RESULTS: Nine patients were infected with serotype 9 S pneumoniae. Four of these patients died; two deaths were directly attributable to the infection. Tested against a battery of haemolytic streptococci and other organisms found in the respiratory tract, only two false positive reactions for PBP 2x were found among S mitis. The method demonstrated that the outbreak strain had altered PBP 1a, 2b, and 2x genes, a pattern clearly distinguishable from other penicillin resistant strains isolated at the same time. CONCLUSIONS: This method is simple to perform and would enable many laboratories to characterise the genotype of penicillin resistant S pneumoniae and investigate transmission in their hospitals.

Burkholderia cepacia: medical, taxonomic and ecological issues.

The increasing challenge posed by multiresistant saprophytes in medical microbiology is strikingly demonstrated by the emergence of Burkholderia (formerly Pseudomonas) cepacia as an opportunist pathogen in immunocompromised patients, particularly individuals with chronic granulomatous disease and cystic fibrosis (CF). Best known previously as a phytopathogen and the cause of soft rot of onions, B. cepacia presents three major problems for the CF community: innate multiresistance to antimicrobial agents; person-to-person transmission of epidemic strains through nosocomial or social contacts; and 'cepacia syndrome', a fulminating fatal pneumonia, sometimes associated with septicaemia, that occurs in approximately 20% of colonised patients, including those with previously mild disease. Accumulated evidence to dispel earlier suggestions that the organism is avirulent and merely a marker of existing lung disease includes: case-controlled studies in CF patients; reports of serious infections in non-CF patients; in-vitro and in-vivo evidence that B. cepacia induces production of pro-inflammatory markers, including the major cytokine TNFalpha; and histopathological evidence that exposure of transgenic CF mice to B. cepacia results in pneumonia. By the early 1990s, the use of selective culture media and DNA-based bacterial fingerprinting confirmed suspicions of epidemic person-to-person spread of B. cepacia. This evidence provided scientific justification for draconian and controversial measures for infection control, in particular, segregation of B. cepacia-colonised patients during treatment at CF centres and their exclusion from social gatherings and national conferences. Recently, molecular analyses of type strains and clinical isolates have revealed that isolates identified previously as B. cepacia belong to at least three distinct species and have increased concern regarding the reliability of current laboratory detection and identification systems. Clarification of the taxonomy of B. cepacia-like organisms and the pathogenic potential of environmental isolates remains a high priority, particularly when the organism's antifungal and degradative properties have created interest in its potential use as a biological control agent to improve crop yields and its use for the bioremediation of contaminated soils.

Dictyostelium discoideum cobB mutants show reduced heavy metal accumulation associated with gene amplification.

Wild-type Dictyostelium discoideum cells growing on non-toxic levels of nickel chloride or cobaltous chloride accumulate 2-3.5 times as much nickel and at least 1.5 times as much cobalt as cobB mutants. The cobB trait is dominant, confers unstable cobalt and nickel resistance and is correlated with the presence of up to 50 copies of a linear extrachromosomal DNA, approximately 100 kb in length, derived from linkage group III. Independent cobB mutants can be obtained by selection on medium containing either cobalt or nickel. The amplified DNA can be transferred to wild-type strains by electroporation. Strains with mutations at a second cobalt resistance locus, cobA, accumulate the same amount of cobalt, but more nickel than wild-type strains. Our results are consistent with the cobA mutant phenotype being due to internal sequestration of cobalt, and the cobB mutant phenotype being due to reduced net uptake of cobalt and nickel. Energy-dependent nickel export was detectable in wild-type and cobB mutant strains but its role in heavy metal resistance has not yet been proved.

The Big-Five factor structure as an integrative framework: an analysis of Clarke's AVA model.

Using a large (N = 3,629) sample of participants selected to be representative of U.S. working adults in the year 2,000, we provide links between the constructs in 2 personality models that have been derived from quite different rationales. We demonstrate the use of a novel procedure for providing orthogonal Big-Five factor scores and use those scores to analyze the scales of the Activity Vector Analysis (AVA). We discuss the implications of our many findings both for the science of personality assessment and for future research using the AVA model.

The replication origin position and its relationship to a negative trans-acting transcription regulator encoded by Dictyostelium discoideum nuclear plasmid Ddp1.

The replication origin of the Dictyostelium discoideum plasmid Ddp1 was localized to a 543-bp region. This includes most of the AT-rich intergenic region between the G1 and G5/D6 genes containing both of their promoters and multiple copies of a TTTTGACT repeat. The G5/D6 gene, which lies adjacent to, and partially overlaps, the 543-bp origin region, encodes a trans-acting factor that negatively regulates transcription of the G4/D5 gene. Inactivation of the G5/D6 gene led to expression of a transcript (G6) 0.2 kb larger than the D5 transcript from the G4/D5 gene in vegetative and developing cells. The G5/D6 gene also regulates transcription of the G1, G2/G3/D4 and G5/D6 genes either alone or in concert with other Ddp1 gene products.

Burkholderia cepacia and cystic fibrosis: do natural environments present a potential hazard?

An environmental survey of 55 sites yielded only 12 Burkholderia cepacia isolates, none of which displayed the phenotypic properties of a multiresistant epidemic strain associated with pulmonary colonization in patients with cystic fibrosis. Although the environment probably poses a low risk for patients with cystic fibrosis as a source of B. cepacia, the pathogenic potential of individual environmental strains remains unclear. We advise caution in the development of B. cepacia as a biocontrol agent.

Plasmid maintenance functions encoded on Dictyostelium discoideum nuclear plasmid Ddp1.

All of the plasmid-carried genes expressed during vegetative growth are essential for long-term maintenance of plasmid Ddp1 in the nucleus of Dictyostelium discoideum. Deletion of Ddp1 genes expressed only during development had no detectable effect on plasmid maintenance. Deletion of vegetatively expressed genes, either singly or in pairs, resulted in (i) a rapid loss of plasmid from cells grown in the absence of selection for plasmid retention, (ii) variation in the proportion of monomer to multimer forms of the plasmid molecules, and/or (iii) abnormalities in plasmid copy number. At least two plasmid-encoded gene products influence patterns of expression of plasmid genes.