ALBERT

Notes: Many new Major Histocompatibility Complex (MHC) genes have been discovered in the last 5 years. Defining the polymorphism of these new genes may elucidate their function and their relevance to diseases with MHC associations. Polymerase chain reaction and single stranded conformation polymorphism (PCR SSCP) analyses were used to detect sequence polymorphisms of PERB1 demonstrated by comparing the available genomic sequence of four haplotypes. This study showed that PCR SSCP of PERB 1 is reproducible. In addition, PERB1 alleles segregate within families together with MHC haplotypes. Typing results from the Forth Asia and Oceania Histocompatibility Workshop (4AOHW) cell panel indicate that the identified polymorphisms of PERB 1 are ‘haplotypic’, i.e., unrelated individuals carrying the same MHC ancestral haplotypes carry the same PERB1 SSCP pattern. Interestingly, PERB1 SSCP patterns allow the distinction of ancestral haplotypes which share HLA-B serological specificities, such as HLA-B44 and therefore this analysis can be used to further define MHC haplotypes and thus to improve our understanding of the evolution of this complex.

Notes: Twenty-eight cases of coeliac disease (CD) and seven of dermatitis herpetiformis (DH) have been verified in Iceland. Standard serological techniques were used for HLA typing. Twenty-five individuals with CD were typed, 21 (84%) of whom carried DR3, DQ2. Twelve of these 25 (48%) had DR3, DR7, DQ2, which makes them possibly homozygous for DQ2, and suggests that homozygosity of DQ2 increases the risk for CD. The four DH patients that were typed all had HLA-B8, DR3, DQ2. It is concluded that CD and DH are associated with DR3, DQZ in Icelanders.

Notes: In order to identify new susceptibility markers for Rheumatoid Arthritis (RA), we analysed the dinucleotide repeat polymorphism at the T cell receptor delta locus (TCRD) in 65 RA patients and 99 healthy Belgian controls. A significant under-representation of the A4-A5 TCRD genotype was observed in the RA population.

Notes: HLA antigens of the Kuwaiti population are not as well characterized as those of other international ethnic groups. We present results for HLA typing of Kuwaiti individuals using commercial Biotest sera. Six hundred and seventy one Kuwaiti were typed for HLA-A, -B, -C antigens and 399 were typed for HLA-DR, -DQ antigens. Antigen frequency and gene frequency were computed for each phenotype observed. The antigens with the highest frequencies were HLA-A2, A1 and A3; B5, B12 and B7; Cw″4 and Cw-l; DR52, DR5, DR3 and DR7; DQ1 and DQ2. HLA haplotypes with strong linkage disequilibrium and characteristic of Kuwaiti population are A2-B5, A1-B8, B7-DR7 and B8-DR3. A comparison study with other populations is presented.

Notes: HLA-B14 positive haplotypes have increased frequencies in a group of patients with puberty disorders, IgA deficiency and cancer of the ovary. Clinical investigations demonstrated that all these patients have high values of 170H progesteron after the ACTH test which suggests an alterated function of 21 hydroxylase enzyme. In order to investigate whether these B14 positive haplotypes carry the same CYP21 mutation in the various diseases and controls, we have amplified by polymerase chain reaction (PCR) the sections of CYP21B gene which include amino acid positions 172 and 281 where typical mutations are known to occur in 21 hydroxylase deficiency. The presence or absence of the defined mutations was tested by oligonucleotide hybridization using oligonucleotides, labelled with DIG-ddUTP, designed to hybridize with the mutated or with the normal sequence. It was found that regardless of whether the subject tested was a patient or a healthy control the mutation at position 281 was found in all cases carrying HLA-B14, DR1 haplotype. Interestingly, this mutation does not seem to be in association with HLA-B14, DR7 haplotype.These findings suggest that CYP21 gene plays a role in all these differing diseases although it must be stressed that there may be alternative explanations for the observed data.

Notes: Patients with CAH, extrahepatic HBV manifestation and healthy children were studied for presence of Gm 1,2,3,10,21 factors and Km 1 factor. Significantly higher frequency of Gm (1, 2, 3, 10, 21) phenotype was shown in CAH group as compared with the other two groups. Relationship between Km factors and examined groups was not found.

Notes: HLA-DRB1, DQA1 and DQB1 alleles have been determined in 42 families with one IDDM proband and 64 healthy controls, by oligotyping (PCR-SSO) using primers and probes from the XI International Histocompatibility Workshop. A positive DRB1 *03 and DRB1 *04 association with the disease was observed, whereas DRB 1*11 and DRB 1 *07 showed negative association but 19% of patients carried DRB1 alleles different to DRB 1 *03 or *04. When single alleles were considered, DQA1 *03 showed the strongest association with susceptibility to the disease (RR = 8.2, Pc = 0.00001) but this association was outgrown by 2 and 3 allele combinations, with genotype DRB 1 *04-DQA 1 *03-DQB1*0302/DRB1*03- DQA 1*0501- DQB 1*0201 showing the strongest association (RR = 28, Pc = 0.002). Application of the relative predispositional effect (RPE) method to our data, revealed a further susceptibility risk provided by the DRB1*13-DQA1*0102-DQB 1*0604 haplotype once DR3 and DR4 haplotypes were removed. When DQA1-DQB1 genotypes were analysed for presence of Arg 52 (DQ α) and absence of Asp 57 (DQ β), genotypes SS/SS were found significantly increased in diabetics. Interestingly, one of the strongest associations with the disease was observed with the DQA 1*03-DQB 1*0201 combination encoded mainly by genes in trans (RR = 11.7 Pc = 0.00004). These observations and their comparison with DR-DQ haplotypes in more homogeneous ethnic groups support the stronger influence of the DQ molecule rather than the individual DR or DQ alleles in the susceptibility to IDDM. They also emphasize the need for detailed HLA haplotype studies in non-Caucasian and ethnically mixed populations to gain further insight into the nature of genetic and environmental factors contribution to autoimmunity.

Notes: The human TNF genes are located within the MHC class-III region on chromosome 6. The presence or absence of an Nco-I restriction site in the 5’ non-coding sequence of the TNFβ gene defines two alleles (TNFB*1 and TNFB*2). The segregation of these alleles has been associated with levels of TNFα or TNFβ production in systemic lupus erythematosis (SLE), insulin-dependent diabetes mellitus (IDDM) and in healthy control individuals.Rheumatoid arthritis (RA) is characterized by high levels of TNFα within the synovial fluid and to address the question of whether this could be brought about by a genetic predisposition to high TNF production by RA individuals, we examined the distribution of this Nco-I polymorphism in 98 healthy volunteers and 123 patients with active rheumatoid arthritis. No difference was observed between the normal and RA groups with respect to haplotype segregation or allelic frequency. Furthermore, no difference was observed between DR4+ or DR4- individuals in the control or RA groups.These data demonstrate that the high level of TNFα seen in the joints of RA patients is unlikely to be due to a genetic predisposition of these patients to high TNFα production, as defined by the TNF Nco-I restriction fragment length polymorphism (RFLP).

Notes: The exceptionally strong independent association found between Lp(a) lipoprotein [Lp(a)] levels and atherosclerotic disorders indicate that Lp(a) is a factor of considerable importance in the pathogenesis of atherosclerosis. The association between Lp(a) and diabetes, rheumatoid arthritis and renal diseases suggest that Lp(a) may be involved in immunological mechanisms.Lp(a) has a great tendency to aggregate and bind to glucosaminoglycans, fibrin and fibronectin and is preferentially retained in the extracellular matrix during development of atherosclerosis and is in vitro phagocytosed by macrophages, probably as small aggregates. It was previously found that the Lp(a) level is significantly related to the HLA class II genotype in male patients with early coronary artery disease. In this paper additional results of interleukin determinations in relation to HLA type and Lp(a) levels are presented and discussed. It is suggested that an autoimmune process, perhaps triggered by a concomitant intracellular infection may occur, especially in patients with inherited high Lp(a) levels in combination with certain inherited HLA class II genotypes.

Notes: DPB1 locus typing of the 155 cell 4AOHW panel was performed using a PCR-RFLP method. Ambiguity of allele assignment was resolved by amplification using sequence-specific primers. Of the 150 cells for which typings were achieved, three exhibited unusual restriction enzyme fragment patterns, suggesting the possibility of novel DPB1 alleles. Sequence analysis revealed one allele present in the currently reported 46, one novel allele (4AOHW/107) not present among the 46, and one from a non-human primate which is being investigated. Twenty-six (26) of the 34 10IHW cells have been studied previously by cDNA RFLP, and strong haplotypic associations have been demonstrated between DPA1 and DPB1 locus alleles. It is proposed that exploitation of intron polymorphisms marking haplotypes will be an integral part of future DPB 1 typing as a ‘first-pass’ stratification process to minimize the requirement for sequence-based methods to definitively assign DPB 1 alleles.

Notes: DNA oligotyping was used to determine HLA-A28 subtypes in 25 unrelated Caucasian individuals living in or around Seville, Spain. Results showed that HLA-A*6802 was the most frequent allele, found in 14 individuals (53.8%), followed by HLA-68.3, which was present in eight subjects (30.8%), and both combined represented 84.6% of A28+ individuals in the area. The HLA-A*6801 allele was found in three individuals (11.5%), whereas HLA-A*6901 was present in one subject only (3.8%). Results indicate that the distribution of HLA-A28 alleles can vary among different Caucasoid populations. In this way, the high frequency obtained for A*6802 supports previous studies suggesting that the HLA-A*6802 allele was prevalent in people of the Mediterranean basin, in contrast to A*6801, prevalent in northern European populations.

Notes: HLA alleles were studied in Kuwaiti patients with Systemic lupus erythematosus (SLE). Although significant association of B5, B8, and DR3 has been reported in the literature, the most common phenotype for our patients is A3, DR2 as susceptible alleles and DQ1 as a protective gene.

Notes: A new HLA-B antigen, HLA-B7Qui that appears to be a variant of HLA-B7 has been identified. This antigen, which is HLA-Bw6 associated, reacts with approximately two-thirds of cytotoxic antisera stimulated by HLA-B7 or B27 that lack a B27 or B7 component, respectively. All anti-B7+27 antisera (stimulated by either B7 or B27) react with B7Qui as do most B22-stimulated sera possessing a B7 component. However, sera stimulated by B60, with or without a B7 component, fail to react with B7Qui.Family studies show the B7Qui allele to be unique to the haplotype –HLA-A32 CW6 B7Qui Bf*S C4A*6 C4B*1 DR11 DQ7 (GL02). Six Caucasoid subjects on the panel of 6861 HLA-typed potential bone marrow donors have this antigen (phenotype frequency, 0.08745%; gene frequency, 0.04473%). Work undertaken during the 11th International Histocompatibility Workshop (Reekers et al., 1992) showed that B7Qui has the same isoelectric point as HLA-B702 and HLA-B703 (Bpot) and that B7Qui is distinct from the HLA-B7 variants B703, B7SL, BDT, B7x40, BRI, and B41v.

Notes: HLA-DRB1*04 allele frequencies have been determined in 184 HLA-DR4-positive unrelated blood donors from the South Wales area, using group-specific polymerase chain reaction (PCR) amplification and hybridization with sequence-specific oligonucleotide probes, PCR amplification with sequence-specific primers, and PCR single-strand conformation polymorphism analysis.Eight of the fifteen known HLA-DR4 sequences were detected in this study. Linkage disequilibrium analysis of HLA-DRB1 *04 and HLA-B, -DR and -DQ alleles revealed distinct haplotypic associations for all the major alleles detected in this population, including the novel linkage of HLA-B55 with DRB1*0407.These results are relevant to the role of HLA-DRB 1*04 haplotypes in determining allogeneic histocompatibility and disease susceptibility.

Notes: A chicken MHC class I (B-F) cDNA from SPAFAS line 11 embryonic liver tissue was isolated and characterized by nucleotide sequencing. Comparing this sequence with previously described B-FcDNAs highlights clustered nucleotide substitutions in exon 3, encoding amino acids located on the a-helical region of the α2 domain.

Notes: Computerized panel training with graphic feedback showed significant feedback effects for magnitude estimation judgments of sweet and sour taste intensities. Context effects still existed but were reduced after training with or without feedback for both line scale and magnitude estimation scaling methods. Following a 2-week period of inactivity, context effects tended to increase. These results suggest implications for current descriptive panel training and maintenance activities.

Notes: Developing peanut breeding strategies for roasted peanut flavor improvement requires that roasting be to an optimum and effects of negative environmental exposure be to a minimum. In practice it is difficult to achieve these conditions. However, statistical adjustments are possible. To ascertain the proper adjustments, surface response equations were determined relating roast color and fruity attribute scores to roasted peanut attribute score. Fruity attribute had a depressing effect on roasted peanut attribute score. Similarly, underroasting as well as overroasting led to reduced roasted peanut attribute scores. Adjusting for genotype and location had nonsignificant effects on mean optimal roast color or linear equation slope values of the fruity-roasted peanut relationship. With these findings a method for maximization of roasted peanut values was developed using a modified SAS routine. Although the capability to estimate the roasted peanut flavor of a sample with optimal roasting and environmental effects has several potential applications, our specific application is to evaluate parentage contribution to flavor enhancement or flavor reduction.

Notes: Sensory terminology is very important in descriptive analysis and perceptions are greatly influenced by the language. Definitions can be very useful for specifying and describing a sensation. However, many sensory attributes are not easily defined and physical reference standards can contribute a great deal to smoothing language problems. They can be used to develop the proper descriptive language, to reduce the amount of time required to train the sensory subjects, and to calibrate the panel in the use of the intensity scale, all the while providing documentation of the sensory terminology.The Quantitative Flavor Profiling (QFP) technique was applied to evaluate flavored cheese analog, yoghurt and sweetened milk samples. Specific standardized flavor languages were developed for each product type and included reference standards for each sensory descriptor. The results of QFP were analyzed by principal component analysis.

Notes: Results obtained in the determination of sensory thresholds with three different simplified methods were compared. The methods were: a modification of the ascending method of limits (1), one based on the method of the constant stimuli (2), and one based on the use of scales (3). The study was carried out on two sensorially simple systems, sucrose and aspartame aqueous solutions. The thresholds values for sucrose obtained by method 1 (1.41 g/L) and by method 2 (1.60 g/L) were similar and within the range obtained by method 3 (0.6–1.6 g/L). For aspartame the values obtained with methods 1 and 2 were also similar (0.004 and 0.005 g/L, respectively), but they were below the minimum value of the corresponding range (0.008–0.016 g/L) calculated with method 3. These results suggest that the sensory methods provoked less variability in thresholds values than other noncontrolled factors. The limitations and advantages of each method are discussed considering: experimental work, precision of results and practical validity.

Notes: In Free Choice Profiling (FCP) each individual panelist creates his own series of descriptors to describe the differences among a set of samples. This is in contrast to multidimensional scaling (MDS) where the panelists assess the similarities among the samples. The objective of this study was to compare the results obtained from two FCP panels (one composed of “sensory-savvy” persons and the other of “sensory-naive” individuals) with MDS results. Vanillin and four vanilla samples each originally at 3-fold, 10-fold and 20-fold concentration were evaluated. On average the sensory-savvy (SS) panelists did not use significantly more descriptors than the sensory-naive (SN) group. However, the terms used by the SS group were more likely to discriminate among the samples than the SN terms. Procrustes analysis indicated that the SN data did not have a true consensus space while the SS data did. The MDS data space was visually similar to the FCP space.

Notes: A well-trained panel is a valuable tool for describing and quantifying characteristics of a food product. This research was undertaken to study the effects of feedback during panel training. A computerized system was designed using the Macintosh computer to gather data and provide panelists with individualized instruction and immediate graphic feedback. Two levels of feedback (with or without) were delivered to the panelists over a 2-week training period. Feedback consisted of correct response for discrimination testing and a graph displaying means and deviations for scaled data.Results showed an expansion in the use of the line scale and an increase in precision across trials. No notable change in magnitude estimation sample scores was observed across feedback conditions; however, deviations were lower following feedback. Although exposure/practice alone provided similar changes, further differences were affected with graphic feedback. Results suggested individualized computer assisted instruction with graphic feedback may provide an efficient and effective tool to complement existing panel training techniques.

Notes: The purpose of this study was to compare how similar results are achieved by three different procedures of measuring liking for ice cream. Subjects (N=56) assessed their liking for three brands of vanilla ice cream on a 9-point scale in seven sessions within three weeks. The examined procedures were (1) tasting and rating all three brands simultaneously side-by-side within the same session, (2) rating each brand in separate sessions after ad libitum consumption and (3) tasting (without consumption) and rating each brand in separate sessions. The liking scores for brands differed significantly but the differences were small because all samples were well-liked. In all procedures, the ratings of liking differed clearly between those who ranked the brand first and those who ranked it second or third, even if there was a considerable number of tied first places. Correlations among the three different measurements of liking were relatively low for all brands of ice cream (r = 0.16 – 0.37). The most preferred brand for most respondents changed from one procedure to another. The perceived characteristics were close to ideal in all brands, and all three procedures gave similar mean results. The distance of mouthfeel, creaminess, sweetness, and vanilla aroma from ideal differed between those who liked the brand best and those who rated it second or third. These differences were larger in side-by-side and after consumption conditions compared to single sample presentation.

Notes: A simple nonmathematical introduction is given to Thurstonian modeling and its application to difference testing. The theoretical treatment explains differences in performance noted for various difference test protocols and the so-called paradox of discriminatory nondiscriminators, whereby a slight change in instructions to the judge can alter the proportion of tests performed correctly. From this, the assumptions in the use of binomial statistics for analyzing difference tests and their shortcomings, are discussed. New ideas on the generality of the paradox of discriminatory nondiscriminators are discussed, along with the effects of the cognitive strategy adopted by the judge during testing.

Notes: An important issue in consumer oriented studies is to measure how closely a specific product or perception matches the representation people have of it. We used a concept matching technique to assess the degree to which a variety of different cheeses matched subjects’ concepts of Cheddar cheese. Eighteen subjects made their judgments using their own past experiences with Cheddar cheeses. They made judgments in three separate conditions: only the odor, only the taste and texture and normal eating. This technique provided valid and reliable information on the extent to which the cheeses matched the subjects’ concept of Cheddar cheese. Agreement between classifications of cheeses based on taste and texture only and based on normal eating was high (R=0.90). Agreement between classifications based only on odor and those based on normal eating was weaker (R-0.59). Thus, the taste and texture of the cheeses were more useful than the odor for classifying cheeses as Cheddar.

Notes: Thirty French red wines, selected from the main regions of France, were chosen as objects for an expert panel to be trained in Descriptive Analysis. Seventeen attributes were chosen to describe the wines. Principal Component analysis showed a good separation of the wines among regions of origin. The results were verified by Analysis of Variance (ANOVA). Generalized Procrustes analysis was included to analyze the panelist performance, such as individual differences in attribute ratings.

Notes: The temporal irritant response was evaluated for six concentrations of capsaicin, cinnamaldehyde, andpiperine, using time-intensity (TI) methodology. TI curves were evaluated using six TI parameters: maximum intensity (Imax), time-to-maximum (Tmax), plateau time (Tplat), total time (Ttot), maximum rate of onset (Monset) and maximum rate of decay (Mdecay) of perception. Maximum intensity was used to evaluate the appropriateness of the Beidler taste equation, calculate the degree of affinity of the stimuli for the receptor (Kb), Gibbs free energy values, and the other TI parameter were used to quantify the adsorption desorptionprocesses. For cinnamaldehyde and capsaicin, correlation coefficients for the proposed Beidler taste model were 0.999, and 0.996, respectively. The large association constants (Kb) for cinnamaldehyde (25 M−1) and capsaicin (5.2 × 104 M−1), compared to taste compounds, were consistent with their steep psychophysical functions and their persistent aftertastes. Concentration dependencies of Tmax Tplat, Ttot, Monset and Mdecay, fit linear and semi-hyperbolic functions and were congruous with the proposed adsorption- desorption model for irritant perception.

Notes: There are two approaches to modeling key relations among variables when one tests products. S-R or stimulus-response modeling assumes that the researcher controls the antecedent physical variables (such as ingredients or processing), and that these physical variables are the primary cause of product-to-product differences. R-R or response-response modeling assumes that the researcher can measure co-varying physical measures of a food, but may or may not have control (or even knowledge) of the antecedent physical variables that generate product differences. S-R modeling allows for true optimization, in terms of defining the operations needed to maximize an attribute (e.g., acceptance). R-R modeling allows only a guess as to what particular combination of physical measures would correspond to a maximum level of the attribute. Often S-R and R-R modeling and optimization are confused with each other, leading to incorrect conclusions.

Notes: Liking rating data of 8 pigmeat products from 99 Australian females aged 25-45 was analyzed using MDPREF. A line scale extended past the anchor points, 100 mm long and anchored by the words ‘dislike extremely’ and ‘like extremely’ was used. Liking was judged on the product name alone, no products were actually tasted. MDPREF configures products and consumers so that individual preferences (or product order preference) can be obtained. The order of product preference from the rating scores and order of product preference from the MDPREF analysis for each consumer was matched. It was found that the preference order for each consumer from the MDPREF map matched the order shown by rating scores in about 25% of cases. MDPREF produced 17% of consumers who had no products in the correct position for the preference order. Only 1% of consumers had all eight product preferences in the correct order.

Notes: Evaluations of multiple products in a category proceed with two different, unstated objectives, and two distinct analytic approaches. The first objective is to “map” the category - viz., to put the products in a category onto some type of geometrical map with the property that products lying close to each other on the map share aspects in common. Researchers use mapping to identify which products compete with each other, as well as to discover whether or not there exist in the category unfilled “holes” that have promise. Researchers use modeling, in contrast, to identify the salient dimensions along which consumers differentiate products in the category. Modeling assumes that the researcher will create a relation between two or more attributes in the category. The analysis generates equations (or even only correlations) rather than maps. Mapping and modeling are complementary, yet often one approach is used to the exclusion of the other because of internal biases and points of view held by the researcher. This paper shows how the two are used, and how they can be integrated to create a more powerful analytic approach.

Notes: This paper proposes a statistical method for difference tests with repetitions. Classical methods for difference tests are based upon the binomial distribution, and are not concerned with the number of repetitions per judge. But when more than one replication of a difference test is required, judgements from different judges are more independent than replicates from the same judge; these two cannot be combined in the classical methods. In this paper, we propose another approach that takes into account two points: the number of repetitions per judge, and the differences within subjects. Two examples are presented to illustrate this approach.

Notes: GRAPES computes individual and global analyses of variance for sensory profiling data, consisting of several sessions in which all the panelists gave scores to all the products for a number of attributes. The fitted model takes into account the session effect. GRAPES summarizes the results by means of graphical assessor scatterplots which allow to check and to compare panelist performances, such as the way of using scale, the reliability, the discrimination power and the agreement with the panel. In addition, GRAPES detects the outliers for each of these criterion. The usefulness of GRAPES for the panel leader will be demonstrated using texture and flavor profiling of 4 restructured steaks by 12 assessors for 15 attributes. The SAS® program GRAPES, available by e-mail from the author, requires the SAS/BASE®, SAS/STAT®, SAS/GRAPH® and SAS/IML® softwares.

Notes: Cheddar cheese (three trials) was made from split lots of curd, salted with NaCl or mixtures of NaCl/KCl and ripened at 3 ± 1C. Preferences for cheese, as determined by consumer panels, were significantly (P 〈 0.05) affected by flavor and order of presentation to panelists but not (P 〉 0.05) by age of cheese. In the first order of presentation, cheese made with NaCl was preferred over those made with NaCl/KCl, whereas in the second order of presentation cheese made with 1.52% NaCl/KCl (2:1) was preferred. In the third order of presentation, cheese made with 1.72% NaCl (rather than 1.5%) was preferred. Overall, mean scores were higher for cheeses made with NaCl than with NaCl/KCl. However, the score of cheese made with ca. 1.5% NaCl/KCl was not significantly (P 〉 0.05) different from that of cheese made with ca. 1.5% NaCl. Cheese made with ca. 1.5% NaCl/KCl (2:1) was consistently preferred over cheeses containing more KCl.

Notes: The responses of Australians and Japanese to the sweetness of 36 products from 6 food categories, namely beverages, biscuits, cereals, chocolates, fruit juices and jams were compared using ‘sweetness liking’, ‘sweetness strength’ and ‘sweetness just right’ scales. Similar responses were obtained for the two panels with jams and fruit juices, but considerable disagreement particularly as regards ‘sweetness liking’ occurred with beverages, biscuits and chocolates where there was evidence that familiarity with the products influenced sweetness responses. Where it was possible to compare the ‘sweetness strength’ of similar products from each country, e.g., strawberry jam, there were no obvious differences. The sweetness of products from both countries covered a wide range of levels, and liking for sweetness levels appeared to be dependent on the context of the product and familiarity with the product. Prediction of liking for the sweetness level of a particular product does not appear possible and it is clear that with a significant number of products from either country that modification of sweetness levels would be necessary for acceptance by the other culture.

Notes: A series of yoghurts was produced by using ten commercially available starter cultures under laboratory conditions and subjected to descriptive sensory analysis. The score sheet was developed by a ten member panel and included eight sensory categories. Additionally, a hedonic scale was used for evaluating acceptability impression. Statistical analysis showed good performance of both individual subjects and the whole panel. Furthermore, homogeneity between replicate assessments was observed. Significant differences between products were found in each sensory category except texture (gel firmness). Correlation analysis showed significant interrelations between some sensory continua. By using multiple regression analysis hedonic scores were found to be mainly determined by ‘Flavor’ and ‘Ropiness’ showing positive and negative weightings, respectively.

Notes: Four combinations of cookie and juice were presented to 42 subjects for ratings of pleasantness of the items and their combinations (Experiment 1). Pleasantness of juice contributed to the pleasantness of a combination more than that of cookie (multiple regression analysis). In Experiment 2, subjects (N=41) rated each combination after ad libitum consumption in four separate sessions. Pleasantness of a cookie contributed more to the pleasantness of a combination than that of juice, and the average R2 obtained in multiple regression analysis was higher than in Experiment 1, suggesting that ratings after ad libitum consumption are more reliable. Pleasantness ratings explained ad libitum consumption of an item up to 23%, but perceived hunger and thirst, and consumption of the other item were at least equally good predictors. It is concluded that the pleasantness is only one among the multiple factors affecting amounts consumed in laboratory conditions.

Notes: Two taste sensitivity measures (detection threshold and time-intensity [T/I]) were applied to evaluate bitter taste sensitivity of caffeine in both caffeine nonusers (n = 12) and users (n=12, caffeine consumption level; 〉 300mg/day). Taste thresholds of caffeine were significantly different between nonusers and users in two test sessions (p 〈 0.01 in first and p 〈 0.05 in second). In T/I measures, only the difference in peak height was observed between the two groups in one of two test sessions. The other two measures, half width and area of T/I curves showed no significant difference.

Notes: This study examined how people compare the hardness of different materials. Simple models with either elastic or plastic behavior were prepared and controlled mechanically. Six elastomers were compared to 29 plastic dental waxes of different hardness. For each elastic sample, a psychophysical staircase method was used to determine its perceived hardness in comparison to the plastic samples. Single bites were performed and the forces were recorded by a small load-cell placed between the sample and the teeth. Nine subjects. free of dental pathology, participated in this study. Subjects were able to match samples of both materials with a specific stress ratio which depended on the hardness of the elastic samples. Results indicated that people do not use the same sensory cues and, in this case, no clear role of the bite force was established.

Notes: The objective of this study was to compare the preferences and performance of three groups of judges, which represented different types and levels of training. Three groups of judges (descriptive analysis judges, ADSA dairy judges and untrained) rated their liking and the sensory attributes of five Cheddar cheeses. The trained panels rated two cheeses relatively lower in liking than did the untrained panel but otherwise generally agreed with the liking ratings of the untrained judges. The trained judges tended to find larger differences in liking among the cheeses than the untrained panel. Training did not improve the agreement among judges on the liking ratings assigned to the cheeses. The trained judges rated the cheeses less intense for most all of the attributes. The three panels disagreed on the relative size and direction of differences in intensity for most all of the attributes. The trained panels did not typically find larger differences among the cheeses in the intensity of the attributes than the untrained panel. Training improved agreement among panel members on the attribute ratings. The attributes of the dairy judges were less intercorrelated.

Notes: Descriptive analysis panelists create, through training, a consensus language to describe perceived differences among samples. If DA gives reliable, objective descriptions of samples, it would be expected that data obtained from independently trained panels be consistent. Two DA panels were trained independently using the same samples. Vanillin and four vanilla samples each at 3-fold, 10-fold and 20-fold concentration were evaluated. Panel J used 14 terms to describe the vanillas and panel K used 16 terms. Eleven and thirteen terms, respectively, significantly discriminated among the samples. Principal component analyses (PCAs) for the two panels were visually similar. Both separated vanillin, Bourbon, Bourbon Processed Bali, Indonesian and Indonesian Non-smoky vanillas across the first PC. Procrustes analysis of the two spaces had a 0.80 fit value. Both the PCAs and the Procrustes analysis indicated considerable overlap of similar descriptive terms. Thus it appears that DA does give reliable consistent results across independently trained panels.

Notes: Odor thresholds in the headspace over aqueous solutions of diacetyl (butane-2,3-dione) were measured in quadruplicate by a forced-choice procedure in 53 individuals. Mean individual thresholds varied over a factor of 256, with the group average at .005 μg/ml. Thresholds showed a modest negative correlation with suprathreshold intensity ratings. Intensity ratings were distributed bimodally, consistent with the existence of insensitive and sensitive groups and suggesting the possibility of a specific anosmia for this compound.

Notes: The relationship between perception of tenderness and chewing activity was explored using the Time-Intensity measurement of perception and electromyographic measurements of masseter muscle movement. Semitendinosus muscles from eight treatments of forage fed beef were evaluated by nine individuals. The Time-Intensity parameters of Area Under the Curve and Decrease Area were the most useful for treatment separation based on tenderness. Information was obtained from the electromyogram regarding the number of chews, time to chew and mastication rate. However, more work with EMG measurements is required to accurately study the effect of masticatory patterns on tenderness perception.

Notes: Graphs useful in exploratory data analysis for checking assumptions and evaluating treatment, sequence, and period effects in a linear model from a crossover design are discussed. In particular, a two-period, two-treatment crossover design of a food sensory study is used to illustrate the applications of these graphs.

Notes: This paper presents a general protocol for the sensory texture profiling of ice cream using eleven descriptors. Four ice cream bases, all containing 10% butter fat, were used in the profiling experiments. The composition of the bases was varied to create differences in softness, cold sensation and toughness. In addition, three sources of error were investigated: temperature, manner of serving and order of presentation. The importance of each type of error was determined by ANOVA techniques. Texture variables (descriptors) are influenced not only by the composition of an ice cream base, but also by the temperature of the ice cream during evaluation. When good standard procedures are used to scoop samples and to present them to panelists, sensory evaluations need not suffer from the other two sources of error examined.

Notes: This paper is about detection of individual differences among assessors in sensory profiling. A discussion of different types of such differences are given and a graphical tool that can be used for detection of some of them is presented. It is also discussed how this tool, the so called Eggshell plot, can be used in practice and how it is related to the different types of individual differences. The theory is illustrated by two examples from sensory profiling, one from analysis of sausages and one from analysis of cheese.

Notes: The quality determining factors of extruded products are affected by the temperature, shear and pressure generated by any input to the extruder during the short residence time (〈 120s). Although the relationship of process history to measurable product qualities has been established, sensory qualities have not been well correlated to these process responses. Sensory attributes of extruded corn meal products were investigated and correlated to measured physical properties in this study. Corn meal was extruded in a twin screw extruder (Baker Perkins MPF 50/25; LD ratio 15:1) with step increases in screw speed from 200-400 rpm, and moisture from 16-22%. Principal component analysis (PCA) of main factors from sensory color, crispness, and adhesiveness was correlated to process torque, pressure and temperature. Spatial distribution of process response and product attributes showed crispness to be dependent on extrusion temperature. Porosity and adhesiveness were not correlated to any measured process response. PCA analysis identified significant differences in the effects of moisture and screw speed input to the extruder on product properties.

Notes: Escherichia coli DnaK, DnaJ and GrpE are required for renaturation of heat-inactivated λ CI857 repressor (Gaitanaris et al., 1990). Here we demonstrate that in addition to the above three proteins, GroEL and GroES are necessary for the CI857 repressor to acquire full activity at the permissive temperature. Although full-length soluble repressor is present at normal amounts, the protein has reduced specific activity and migrates abnormally on native gels. To determine where the different chaperones act in protein folding, we identified their cellular locations. DnaK and DnaJ are associated with nascent polypeptide chains in translating ribosomes. In contrast, GroEL, although it is transiently associated with newly synthesized proteins, is absent from the ribosomes. This suggests that DnaK and DnaJ ptay an early role in protein maturation, whereas GroEL acts at a later stage.

Notes: A method of insertional mutagenesis for naturally transformable organisms has been adapted from Haemophilus influenzae and applied to the study of the pathogenesis of Campylobacter jejuni. A series of kanamycin-resistant Insertional mutants of C. jejuni 81–176 has been generated and screened for loss of ability to invade INT407 cells. Eight noninvasive mutants were identified which showed 18-200-fold reductions in the level of invasion compared with the parent. Three of these eight show defects in motility, and five are fully motile. The three mutants with motility defects were further characterized to evaluate the method. One mutant, K2–32, which is non-adherent and non-invasive, has an insertion of the kanamycin-resistance cassette into the flaA flagellin gene and has greatly reduced motility and a truncated flagellar filament typical of flaA mutants. The adherent non-invasive mutants K2–37 and K2–55 are phenotypically paralysed, i.e. they have a full-length flagellar filament but are non-motile. All three mutants show an aberration in flagellar structure at the point at which the filament attaches to the cell. Mutants K2–37 and K2–55 represent overlapping deletions affecting the same gene, termed pflA (paralysed flagella). This gene encodes a predicted protein of 788 amino acid residues and a molecular weight of 90 977 with no significant homology to known proteins. Site-specific insertional mutants into this open reading frame result in the same paralysed flagellar phenotype and the same invasion defects as the original mutants.

Notes: Aspergillus fumigatus secretes a serine alkaline protease (ALP) and a metalloprotease (MEP) when the fungus is cultivated in the presence of collagen as sole nitrogen and carbon source. The gene encoding ALP was isolated and characterized previously. We report here the cloning and the sequencing of the gene encoding MEP. Genomic and cDNA clones were isolated from A. fumigatus libraries using synthetic oligonucleotides as probes. Stretches of the deduced amino acid sequence were found to be in agreement with the N-terminal amino acid sequence of MEP and with internal peptide sequences. The amino acid sequence of the enzyme contains a putative active-site sequence HEYTH homologous to the active site of other bacterial and eukaryotic zinc metalloproteases. Sequence analysis reveals that MEP has a pre-proregion consisting of 245 amino acid residues preceding the 388 amino acid residues of the mature region (molecular mass of 42 kDa). An alp mep mutant, deficient in proteolytic activity at neutral pH in vitro, was constructed and tested for pathogenicity in a murine model. No difference in pathogenicity was observed between the wild-type strain and the alp mep double mutant, suggesting that ALP and MEP are not essential for the invasion of the lung tissues by A. fumigatus.

Notes: An alkali-sensitive mutant, 38154, of the alkalophilic Bacillus sp. strain C-125 could not grow at an alkaline pH. The nucleotide sequence of a 3.7 kb parental DNA fragment that recovers the growth of 38154 at alkaline pH has four open reading frames (ORF1–4). By sub-cloning the fragment, we demonstrated that a 0.25 kb DNA region is responsible for the recovery. Direct sequencing of the mutant's corresponding region revealed a G to A substitution. The mutation resulted in an amino acid substitution from Gly-393 to Arg of the putative 0RF1 product, which was deduced to be an 804-amino-acid polypeptide with a molecular weight of 89 070. The N-terminal part of the putative ORF1 product showed amino acid similarity to those of the chain-5 products of eukaryotic NADH quinine oxidoreductases. Membrane vesicles prepared from 38154 did not show membrane potential (δψ)-driven Na+/H+ antiporter activity. Antiporter activity was resumed by introducing a parental DNA fragment which recovered the mutant's alkalophily. These results indicate that the mutation in 38154 affects, either directly or indirectly, the electrogenic Na+/H+ antiporter activity. This is the first report which shows that a gene responsible for the Na+/H+ anti-porter system is important in the alkalophily of alkalo-philic microorganisms.

Notes: All Helicobacter pylori isolates synthesize a 54 kDa immunodominant protein that was reported to be associated with the nickel-dependent urease of H. pylori. This protein was recently recognized as a homologue of the heat-shock protein of the GroEL class. The gene encoding the GroEL-like protein of H. pylori (HspB) was cloned (plLL689) and was shown to belong to a bicistronic operon including the hspA and hspB genes. In Escherichia coli. the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion element that mapped upstream to the two open reading frames (ORFs). IS5 was absent from the H. pylori genome, and was thus acquired during the cosmid cloning process. hspA and hspB encoded polypeptides of 118 and 545 amino acid residues, corresponding to calculated molecular masses of 13.0 and 58.2 kDa, respectively. Amino acid sequence comparison studies revealed that, although H. pylori HspA and HspB proteins were highly similar to their bacterial homologues, the H. pylori HspA featured a striking motif at the C-terminus. This unique motif consists of a series of cysteine and histidine residues resembling a nickel-binding domain, which is not present in any of the other bacterial GroES homologues so far characterized. When the plLL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (plLL763) into an E. coli host strain, an increase of urease activity was observed. This suggested a close interaction between the HspA and HspB proteins and the urease enzyme, and a possible role for HspA in ihe chelation of nickel ions. The genes encoding each of the HspA and HspB polypeptides were cloned, expressed independently as proteins fused to the maltose-binding protein (WIBP) and purified in large scale. The MBP-HspA and MBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H. pylori-infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic as proteins fused to the maltose-binding protein (WIBP) and purified in large scale. The MBP-HspA and WlBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H, py/or/-infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic.

Notes: In most soft-rotting Erwinia spp., including E. carotovora sub sp. carotovora strain 71 (Ecc71), production of the plant cell wall degrading enzyme pectin lyase (PnI) is activated by DNA-damaging agents such as mitomycin C (MC). Induction of PnI production in Ecc71 requires a functional recA gene and the rdg locus DNA sequencing and RNA analyses revealed that the rdg locus contains two regulatory genes, rdgA and rdgB, in separate transcriptional units. There is high homology between RdgA and repressers of lambdoid phages, specially φ80. RdgB, however, has significant homology with transcriptional activators of Mu phage. Both RdgA and RdgB are also predicted to possess helix-turn-helix motifs. By replacing the rdgB promoter with the IPTG-inducible tac promoter, we have determined that rdgB by itself can activate PnI production in Escherichia coli. However, deletion analysis of rdg+ DNA indicated that, when driven by their native promoters, functions of both rdgA and rdgB are required for the induction of pnIA expression by MC treatment. While rdgB transcription occurs only after MC treatment, a substantial level of rdgA mRNA is detected in the absence of MC treatment. Moreover, upon induction with MC, a new rdgA mRNA species, initiated from a different start site, is produced at a high level. Thus, the two closely linked rdgA and rdgB genes, required for the regulation of PnI production, are expressed differently in Ecc71.

Notes: The PilR protein of Pseudomonas aeruginosa is a transcriptional activator of the pilin gene and belongs to a two-component sensor–regulator family. PilR was overproduced by fusing pilR to the gene for the maltose-binding protein (malE), yielding a MalE–PilR hybrid protein. The plasmid with the malE–pilR fusion, when introduced into a non-piliated pilR mutant strain of P. aeruginosa, restored piliation, indicating that the hybrid protein retains PilR function in vivo. The MalE-PilR protein was purified from Escherichia coli and used in a series of DNA-binding studies. A specific pilin promoter-binding activity of MalE-PilR was observed in a gef retardation assay. Subsequent DNase I footprinting analysis revealed a 40bp PilR-binding site located at the −120 to −80 region, relative to the transcriptional start site of the pilin gene. This PilR-binding region consists of a nine-base sequence and three consensus sequences of 5-(N)4–6C/GTGTC-3′, in a tandem array in which the first 7–9 bp are bound by the PilR on the non-Goding strand, leaving the last two nucleotides (TC) unbound. On the coding strand, PilR binds of sequences complementary to the two middle consensus sequences of the non-coding strand. A sequence similar to the NifA recognition site (5-TGT-(N)11-ACA-3′) is also found within the PilR-binding region. Deletion analysis and disruption of the individual consensus PilR-binding sequences by site-directed mutagenesis revealed that all four PilRbinding sites are absolutely required for the PilS/PilR-mediated pilin gene expression. The presence of four PilR-binding repeat sequences suggests that PilR protein may bind co-operatively or as a multimer.

Notes: The identical partial deep-core structure of Hepα1–3Hepα1–5KDO In Salmonella typhimurium LT2 LPS and Neisseria gonorrhoeae LOS enabled us to isolate a DNA fragment from N. gonorrhoeae that was able to complement the α1,5 LOS heptosyltransferase defect in the S. typhimurium rfaC630 (SA1377) mutant. SDS-PAGE analysis confirmed the production of wild-type LPS in the transformant. Subcloning revealed that complementation was due to a 1.2 kb fragment. Sequence analysis revealed a complete open reading frame capable of encoding a 36–37 kDa peptide. In vitro transcription-translation analysis of the 1.2 kb clone confirmed that a 37 kDa protein was encoded by this DNA fragment. The DNA sequence-deduced protein had 36% identity and 58% similarity to S. typhimurium heptosyltransferase I (RfaC). Primer extension analysis indicated that transcription of the cloned gene in N. gonorrhoeae strain 1291 begins 144bp upstream of the start codon at a G nucleotide. An isogenic mutant of N. gonorrhoeae strain 1291 with an m-Tn3 insertion inside the coding sequence expressed a single truncated LOS with a similar molecular mass to S. typhimurium rfaC LPS. We conclude that the 1.2 kb fragment encodes the α1,5 LOS heptosyltransferase 1 (RfaC) in N. gonorrhoeae. Our studies also provide further evidence that the third KDO residue in S. typhimurium LPS is added after the core synthesis is completed.

Notes: Earlier work has shown that the afsR genetic locus promotes formation of the pigmented antibiotics actinorhadin and undecylprodiglosin in Streptomyces lividans and its close relative, Streptomyces coeiicolor. A protein designated as AfsR has been implicated in this activity. We report here the existence of a previously unknown gene, afsR2, which is separate from and adjacent to the AfsR-encoding sequence and which, when present at high copy number, (i) stimulates transcription of biosynthetic and regulatory genes in the actinorhodin gene cluster (act), and (ii) stimulates the synthesis of undecyiprodigiosin., We show that the effects of afsR2 on actinorhodin synthesis are mediated through transcription of the actll-0RF4 locus, which encodes a transcriptional activator of other genes in the act cluster. Analysis of the oloned afsR2 gene indicates that its activity is the result of the 63-amino-acid protein it specifies.

Notes: Two repeated DNA elements of 103 bp and 105 bp were discovered in brucellae and designated Bru-RS1 and Bru-RS2, respectively. The two elements are palindromic, are 65% similar in sequence, form two families of elements that are slightly divergent in sequence, appear to be intergenic, and are found, collectively, in more than 35 copies in brucellae. These elements are bounded by perfect or nearly perfect inverted repeats. A third copy of the terminal repeat is found within the elements and is the terminus for several truncated copies of the Bru-RS1 family. Hybridization patterns for the elements among brucellae were unique. The elements are dispersed, highly conserved among brucellae, and hot-spots for insertion by IS711.

Notes: The genes for a new type of a haem-copper cytochrome oxidase were cloned from Rhodobacter capsulatus strain 37b4, using the Bradyrhizobium japonicum fixNOQP gene region as a hybridizing probe. Four genes, probably organized in an operon (ccoNOQP), were identified; their products share extensive amino acid sequence similarity with the FixN, O, Q and P proteins that have recently been shown to be the subunits of a cb-type oxidase. CcoN is a c-type cytochrome, CcoO and CcoP are membrane-bound mono- and dihaem c-type cytochromes and CcoQ is a small membrane protein of unknown function. Genes for a similar oxidase are also present in other non-rhizobial bacterial species such as Azoto-bacter vinelandii, Agrobacterium tumefaciens and Pseudomonas aeruginosa, as revealed by polymerase chain reaction analysis. A ccoN mutant was constructed whose phenotype, in combination with the structural information on the gene products, provides evidence that the CcoNOQP oxidase is a cytochrome c oxidase of the cb type, which supports aerobic respiration in R. capsulatus and which is probably identical to the cbb3-type oxidase that was recently purified from a different strain of the same species. Mutant analysis also showed that this oxidase has no influence on photosynthetic growth and nitrogen-fixation activity.

Notes: The human oral bacterium Streptococcus gordonii expresses, on the cell surface, two antigenically related high-molecular-mass polypeptides denoted CshA and CshB, encoded by genes at separate chromosomal loci. The precursor form of CshA is composed of four distinct segments: (i) a 41-amino-acid residue leader peptide, (ii) W-terminal 42–878 residues, (iii) residues 879–2417 comprising 13 repeat blocks of 101 amino acid residues and three shorter blocks, and (iv) a C-terminal anchor domain similar to those present in some other Gram-positive bacterial cell-wall polypeptides. Insertional mutations within cshA reduced both cell-surface hydrophobicity and ability to adhere to oral Actinomyces naeslundii. Insertional mutations in cshB had less effect on hydrophobicity and coadherence. However, expression of both polypeptides was found to be necessary for streptococci to colonize the murine oral cavity.

Notes: The polymerase activity of DNA polymerase I is important for the establishment of the pLS1 replicon by reconstitutive assembly in Streptococcus pneumoniae after uptake of exogenous pLS1 plasmid DNA. In polA mutants lacking the polymerase domain, such establishment was reduced at least 10-fold in frequency. Chromosomally facilitated establishment of pLS1-based plasmids carrying DNA homologous to the host chromosome was not so affected. However, both types of plasmid transfer gave mostly small colonies on initial selection, which was indicative of a defect in replication and filling of the plasmid pool. Once established, the pLS1-based plasmids replicated in polA mutants, but they showed segregational instability. This defect was not observed in strains with the wild-type enzyme or in an S. pneumoniae strain that encodes the polymerase and exonuclease domains of the enzyme on separate fragments. The role of DNA polymerase I in stably maintaining the plasmids depends on its polymerizing function in three separate steps of rolling-circle replication, as indicated by the accumulation of different replication intermediate forms in polA mutants. Furthermore, examination of the segregational stability of the pLS1 replicon in an Escherichia coli mutant system indicated that both the polymerase and the 5′-to-3′ exonuclease activities of DNA polymerase I function in plasmid replication.

Notes: Myxococous xanthus cells can glide both as individual cells, dependent on Adventurous motility (A motility), and as groups of cells, dependent upon Social motility (S motility), Tn5-lac mutagenesis was used to generate 16 new A- and nine new S- mutations. In contrast with previous results, we find that subsets of A- mutants are defective in fruiting body morphogenesis and/or myxospore differentiation. All S- mutants are defective in fruiting body morphogenesis, consistent with previous results. Whereas some S- mutants produce a wild-type complement of spores, others are defective in the differentiation of myxospores. Therefore, a subset of the A genes and all of the S genes are critical for fruiting body morphogenesis. Subsets of both A and S genes are essential for sporulation. Three S::Tn5–lac insertions result in surprising phenotypes. Colonies of two S- mutants glide on ‘swim’ (0.35% agar) plates to form fractal patterns. These S- mutants are the first examples of a bacterium in which mutations result in fractal patterns of colonial spreading. An otherwise wild-type strain with one S- insertion resembles the frz- sglA1- mutants upon development, suggesting that this S- gene defines a new chemotaxis component in M. xanthus.

Notes: The tylosin producer Streptomyces fradiae contains four known resistance genes, two of which (tlrA and tlrD) encode methyltransferases that act on ribosomal RNA at a common site. Expression of tlrA is regulated via transcriptional attenuation. A short transcript, only 411 nucleotides long, terminates 27 nucleotides into the methylase-coding sequence in the uninduced state. Induction of tlrA is proposed to involve a ribosome-mediated conformational change within the mRNA leader that allows transcription to continue beyond the attenuation site, resulting in a transcript about 1450 nucleotides long. Transplantation of tlrD and/or tlrA into Streptomyces albus revealed that the induction specificity of tlrA depends upon the state of the ribosomes and is significantly altered in strains also expressing tlrD.

Notes: Pyelonephritic isolates of Escherichia coli commonly express P-pili, which mediate bacterial attachment to glycolipids on epithelial cell surfaces. Three classes of P-pili have been defined, based on varying specificity for galabiose-containing glycolipids. Variation in adhesive capacity is correlated with a shift in preferred host, suggesting that host tropism depends largely on detailed specificity for the globoseries glycolipids. In this study we examined the importance of the PapG adhesin in determining receptor specificity. Translational fusions were constructed between the ammo-terminus of the PapG adhesin from each of the three pilus classes and a reporter protein. The binding specificity of the purified fusion proteins in vitro was identical to that seen with whole bacteria. Adherence of intact bacteria to cultured kidney cells was markedly reduced by a monoclonal antibody specific for the Class III adhesin (previously denoted PrsG), confirming the importance of the ammo-terminus of PapG in mediating attachment to a receptor when presented on the eukaryotic cell surface. These results suggest that the detailed receptor specificity resides solely within the amino-terminus of the PapG adhesin and is independent of the complex pilus architecture.

Notes: pAMβ1 is a low-copy-number, promiscuous plasmid from Gram-positive bacteria that replicates by a unidirectional theta-type mode. Its replication is initiated by an original mechanism, involving the positive rate-limiting RepE protein. Here we show that the pAMβ1-encoded CopF protein is involved in negative regulation of the plasmid copy number. CopF represses -10-fold the transcription initiated at the promoter of the repE gene and binds to a 31 bp segment which is located immediately upstream of the -35 box of the repE promoter. We propose that CopF inhibits initiation of transcription at the repE promoter by binding to its operator.

Notes: Development of the Escherichia coli cell division site was studied in wild-type cells and in non-septate filaments of ftsZ null and ftsZTs mutant cells. Localized regions of plasmolysis were used as markers for the positions of annular structures that are thought to be related to the periseptal annuli that flank the ingrowing septum during cytokinesis. The results show that these structures are localized at potential division sites in non-septate filaments of FtsZ- cells, contrary to previous reports. The positions of the structures along the long axis of the cells in both wild-type cells and FtsZ- filaments were unaffected by the presence of plasmolysis bays at the cell poles. These results do not agree with a previous suggestion that the apparent association of plasmolysis bays with future division sites was artefactual. They support the view that division sites begin to differentiate before the initiation of septal ingrowth and that plasmolysis bays and the annular attachments that define them, mark the locations of these early events in the division process.

Notes: Monoclonal antibodies were raised against a fusion between the Escherichia coli maltose-binding protein and LciA, the immunity protein that protects Lactococcus lactis against the effects of the bacteriocin lactococcin A. One of the antibodies directed against the LciA moiety of the fusion protein was used to locate the immunity protein in the L. lactis producer cell. LciA was present in the cytosolic. the membrane-associated, and the membrane fractions in roughly equal amounts, irrespective of the production by the cells of lactococcin A.The monoclonal antibody specifically reacted with right-side-out vesicles obtained from a strain producing the immunity protein. It did not react with inside-out vesicles of the same strain, or with right-side-out vesicles obtained from a strain producing both LciA and lactococcin A. Also, externally added lactococcin A blocked the interaction between the antibody and right-side-out vesicles obtained from a strain producing only LciA.The epitope in LciA was localized between amino acid residues 60 and 80. As the epitope could be removed from right-side-out vesicles by proteinase K, it is located at the outside of the cell.The immunity protein contains a putative a-amphiphilic helix from residue 29 to 47. A model is proposed in which this helix is thought to traverse the membrane in such a way that the C-terminal part of the protein, containing the epitope, is on the outside of the cell.Vesicle-fusion studies together with leucine-uptake experiments suggest that the immunity protein interacts with the putative receptor for lactococcin A, thus preventing pore formation by the bacteriocin.

Notes: The rfbkpO1 gene cluster of Klebsiella pneumoniae O1 directs synthesis of the D-galactan I component of the lipopolysaccharide O-antigen. The first two genes in the rfbkpO1cluster encode RrfbkpO1and RfbBKpO1, with predicted sizes of 29.5 or 30.0 kDa and 27.4 kDa, respectively. RfbBKpO1 contains a consensus ATP-binding domain and shares homology with several proteins which function as ATP-binding components of cell surface polysaccharide transporters. RfbAKpO1 is predicted to be an integral membrane protein with five putative membrane-spanning domains and its transmembrane topology was confirmed by TnphoA mutagenesis. The hydropathy plot of RfbAKpO1 resembles KpsM, the transcytoplasmic membrane component of the capsular polysaccharide transporter from Escherichia coli K-1 and K-5. These relationships suggest that RfbAKpO1 and RfbBKpO1 belong to a family of two-component ABC (ATP-binding cassette) transporters. E. coli K-12 containing a plasmid carrying an rfbKpO1 gene cluster deleted in rfbAKpO1 and rfbBKpO1 expresses rough lipopolysaccharide molecules on its surface and accumulates cytoplasmic O-antigen. When RfbAKpO1 and RfbBKpO1 are supplied in trans by a compatible plasmid, O-polysaccharide transport is restored and smooth D-galactan l-substituted lipopolysaccharide is produced. RfbAKpO1 and RfbBKpO1 are, therefore, proposed to constitute a system required for transport of D-galactan I across the cytoplasmic membrane, where RfbAKpO1 represents the membrane-spanning translocator and RfbBKpO1 couples the energy of ATP hydrolysis to the transport process.

Notes: Non-transmissible derivatives of the Streptomyces multi-copy plasmid pIJ101 were mobilized, by co-integrate formation, at frequencies approaching 100% (measured per recipient) by derivatives of the conjugative, low-copy-number Streptomyces coeli-color A3(2) plasmid SCP2*. Efficient co-integrate formation required that the plasmids shared at least 112 bp sequence identity, and it occurred only during conjugation. An SCP2* plasmid gene is involved in the process. Co-integrates were presumably formed in the donor cells and transported to the recipient cells. This is a new phenomenon, not known in other bacteria.

Notes: Integration of plasmid pCGL320 into a Corynebacterium glutamicum ATCC21086 derivative led to tandem amplification of the inserted plasmid (Labarre et a/., 1993). One amplification event was associated with integration of an insertion sequence that we have named IS 1206. Hybridizing sequences were only found in C. glutamicum strains and at various copy numbers. IS1206 is 1290bp long, carries 32 bp imperfect inverted repeats and generates a 3bp duplication of the target DNA upon insertion. IS1206 presents the features characteristic of the IS3 family and part of the DNA sequence centering on the putative transposase region (orfB) is similar to those of IS3 and some other related elements. Phylogenetic analysis of orfB deduced protein sequences from IS 1206 and IS3-related elements contradicts the phylogeny of the species, suggesting that evolution of these elements might be complex. Horizontal transfer could be invoked but other alternatives like ancestral polymorphism or/and different rates of evolution could also be involved.

Notes: Disulphides are often vital for the folding and stability of proteins. Dedicated enzymatic systems have been discovered that catalyse the formation of disulphides in the periplasm of prokaryotes. These discoveries provide compelling evidence for the actual catalysis of protein folding in vivo. Disulphide bond formation in Escherichia coli is catalysed by at least three ‘Dsb’ proteins; DsbA, -B and -C. The DsbA protein has an extremely reactive, oxidizing disulphide which it simply donates directly to other proteins. DsbB is required for the reoxidation of DsbA. DsbC is active in disulphide rearrangements and appears to work synergistically with DsbA. The relative rarity of disulphides in cytoplasmic proteins appears to be dependent upon a disulphide-destruction machine. One pivotal cog in this machine is thioredoxin reductase.

Notes: Campylobacter jejuni is a significant cause of bacterial enteritis in humans. Three of seven C. jejuni isolates examined were found to contain fragmented 23S rRNA. The occurrence of fragmented 23S rRNA correlated with the presence of an intervening sequence (IVS) within the 23S rRNA genes. The IVS is 157 nucleotides in length and replaces an eight nucleotide sequence in the 23S rRNA genes of C. jejuni isolates that contain intact 23S rRNA. The two ends of the IVS share 31 bases of complementarity that could form a stem-loop structure. Fragmentation of the 23S ribosomal RNA results from the excision of the IVS from the transcribed RNA; the 3’ cleavage site maps within the putative stem-loop formed by the IVS. Southern hybridization analysis revealed that the IVS is not present in the genomes of isolates that contain intact 23S rRNA, suggesting that the IVS is not derived from Campylobacter chromosomal sequences. The C. jejuni IVS is located at a position analogous to that of the IVSs found in both Salmonella and Yersinia spp.

Notes: We have studied the formation of spontaneous mutations on plasmids present In the monomeric and dimeric states in a recF strain of Escherichia coli. Two test systems were employed: (i) the precise excision of Tn5 from the tetA gene of the plasmid pBR322 and (ii) operator constitutive (Oc) mutations on the pBR322-derived plasmid pPY97. The rate of Oc mutations was increased by a factor of three when this plasmid was present in the dimeric state compared to the monomeric state and the Oc phenotype was caused by small deletions in the operator sequence. No apparent mutational hot-spot was found. The rate of Tn5 excision was increased on dimeric compared to monomeric plasmids. Excision from a dimeric plasmid usually resulted in two types of mutant plasmids; a dimeric plasmid, where the Tn5 had excised from one of the plasmid units, and a monomeric parental pBR322. A mechanism to account for this is suggested. Complementation tests revealed that the increased mutation rate on dimeric plasmids is the result of dimers being mutaphilic per se, rather than the result of a general, trans-acting increase in mutation rates of the host, induced by the presence of the dimeric plasmid. Furthermore, it was found that the rate of Tn5 excision from plasmids in the monomeric state was increased when the region carrying the inserted Tn5 was duplicated.

Notes: A 3.2 kb EcoRI genomic DNA fragment of Coxiella burnetii was isolated by virtue of Its ability to suppress mucoidy in Eschertchia coli. Nucleotide sequence analysis revealed the presence of the genes homologous to rnc, era and recO of E. coli. Suppression of capsule synthesis, measured by β-galactosidase expression in Ion cps-lac fusion strains of E. coli, is caused by gene-dosage effects of the plasmid-borne rnc genes of either C. burnetii or E. coli. The rnc gene of C. burnetii complemented rn– E. coli hosts for lambda plaque morphology and stimulation of lambda N gene expression. We also demonstrated heterologous complementation of an E coli strain defective for the expression of Era, an essential protein in E. coli, using the plasmid-borne C. burnetii era. Under the control of the bacteriophage lambda PL promoter, this 3.2 kb EcoRI DNA fragment directed the synthesis in E. coli of three proteins with approximate molecular masses of 35,27 and 25 kDa. Antibodies against purified E. coli Era protein cross-reacted with the 35 kDa protein of C. burnetii on Western blots.

Notes: Aspergillus nidulans reproduces asexually by forming thousands of mitotically derived spores atop highly specialized multicellular organs termed conidiophores. We have identified a gene called flbA (for fluffy low brlA expression) that is required for initiation of A. nidulans conidiophore development. flbA mutants form abnormal colonies that have a distinct fluffy phenotype characterized by tightly interwoven aerial hyphae that autolyse as the colony matures. The requirement for fIbA in conidiophore development precedes activation of brlA, a primary regulator of conidiophore development. The wild-type flbA gene was isolated and found to encode a 3.0 kb mRNA that is expressed throughout the A. nidulans asexual life cycle. Overexpression of fIbA using an Inducible promoter resulted in misscheduled expression of brlA in vegetative ceils and caused hyphal tips to differentiate into spore-producing structures. Sequence analysis of a nearly full-length fIbA cDNA clone showed that fibA is predicted to encode a 717-amino-acid polypeptide with 30% identity to the Saccharomyces cerevisiae SST2 protein. SST2 is required by yeast cells for resuming growth following prolonged exposure to yeast mating pheromone and for mating partner discrimination. We propose that fIbA plays a related role in a signalling pathway for Aspergillus conidiophore development.

Notes: In the course of studies on anaerobic citrate metabolism in Klebsiella pneumoniae, the DNA region upstream of the gene for the sodium-dependent citrate carrier (dtS) was investigated. Nucleotide sequence analysis revealed a cluster of five new genes that were oriented inversely to citS and probaby form an operon. The genes were named citCDEFG. Based on known protein sequence data, the gene products derived from citD, citE and citF could be identified as the λ-, β-, and α-subunits of citrate lyase, respectively. This enzyme catalyses the cleavage of citrate to oxaloacetate and acetate. The gene product derived from citC (calculated Mr 36476) exhibited no obvious similarity to other proteins. In the presence of acetate and ATP, cell extracts from a citC-expressing Escherichia coli strain were able to reactivate purified citrate lyase from K. pneumoniae that had been inactivated by chemical deacetylation of the prosthetic group. This represents 5-phosphoribosyi-dephospho-acetyl-coenzyme A which is covalently bound to serine-14 of the acyl carrier protein (λ-subunit). CitC was thus identified as acetate:SH-citrate lyase ligase. The function of the gene product derived from citG (Mr 32 645) has not yet been identified. Expression of the CitCDEFG gene cluster in E. coli led to the formation of citrate lyase which was active only in the presence of acetyl-coenzyme A, a compound known to substitute for the prosthetic group. These and other data strongly indicated that the enzyme synthesized in E. coli lacked its prosthetic group. Thus, additional genes besides citCDEFG appear to be required for the formation of holo-citrate lyase.

Notes: Neisseria gonorrhoeae homologues of gyrA and parC have been identified using hybridization probes generated from conserved regions of diverse gyrA genes. These genes have been tentatively identified as gyrA and parC, based on predicted amino acid sequence homologies to known GyrA homologues from numerous bacterial species and to ParC from Escherichia coli and Salmonella typhimurium. The gyrA gene maps to a physical location distant from the gyrB locus on the gonococcal chromosome, which is similar to the situation found in E. coli. The parC gene is not closely linked (i.e. greater than 9 kb) to an identifiable parE gene in N. gonorrhoeae. The gonococcal GyrA is slightly larger than its E. coli homologue and contains several small insertions near the O-terminus of the predicted open reading frame. A series of ciprofloxacin-resistant mutants were selected by passage of N. gonorrhoeae on increasing concentrations of the antibiotic. Sequential passage resulted in the selection of isolates with minimum inhibitory concentrations approximately 10000-fold higher than the parental strain. Mutations within gyrA resulted in low to moderate levels of resistance, while strains with high-level resistance acquired analogous mutations in both gyrA and parC. Resistance mutations were readily transferred between N. gonorrhoeae strains by transformation. The frequencies of transformation, resulting in different levels of ciprofloxacin resistance, further support the notion that both gyrA and parC genes are invoived in the establishment of extreme levels of ciprofloxacin resistance.

Notes: Co-ordinate expression of many virulence genes in Vibrio cholerae is under the control of the ToxR and ToxT proteins. These proteins function in a regulatory cascade in which ToxR is required to activate toxT, and ToxT activates virulence genes. The precise mechanism for ToxR activation of toxT is unknown, but data presented in this report suggest a direct involvement of ToxR. Primer extension and gene fusion analyses identified a ToxR-regulated promoter directly upstream of toxT, immediately following a region of inverted repeats capable of terminating transcription. Gel mobility shift studies indicate that ToxR binds DNA within the inverted repeat region, yet preliminary evidence suggests that ToxR binding alone is not sufficient for activation of toxT. Possible mechanisms of ToxR-dependent toxT expression are discussed.

Notes: The Escherichia coli CadC protein is required for activation of cadBA transcription under conditions of low external pH and exogenous lysine. cadBA encodes proteins involved in the decarboxylation of lysine to cadaverine, and cadaverine excretion. Sequence analysis suggested that CadC contains a single transmembrane segment separating a DNA-binding domain in the amino terminus from a periplasmic domain. Western analysis of subcellular fractions demonstrated that CadC is expressed and concentrated in the cytoplasmic membrane in cells grown either at an inducing pH (pH5.8) or at a non-inducing pH (pH7.6.). Eight cadC mutants were isolated based on their ability to confer expression of a cadA-lacZ fusion independent of low external pH or exogenous lysine. Five of these mutants expressed the cadA-lacZ fusion at both pH 5.8 and pH 7.6, but retained the requirement for the lysine signal while the other three mutants displayed pH independence in the presence of lysine, and lysine independence at pH 5.8 but not at pH 7.6. These results support a model in which CadC is a membrane-bound transcriptional activator of the cadBA operon capable of sensing (directly or indirectly) signals generated outside the cytoplasmic membrane as a consequence of acidic pH and lysine.

Notes: Computer analysis of the crystallographic structure of the A subunit of Escherichia coil heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53 → Glu or Asp, Ser-63 → Lys, Val-97 → Lys, Tyr-104 → Lys or Asp, and Ser-14 → Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41 → Phe, Ala-45 → Tyr or Glu, Val-53 → Tyr, Val-60 → Gly, Ser-68 → Pro, His-70 → Pro, Val-97 → Tyr and Ser-114 → Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54 → Lys or Ala, Tyr-59 → Met, Ser-68 → Lys, Ala-72 → Arg, His or Asp and Arg-192 → Asn. The results provide a further understanding of the structure–function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.

Notes: Many surface proteins are thought to be anchored to the cell wall of Gram-positive bacteria via their C-terminus. Cell wall anchoring requires a specific sorting signal, normally located at the predicted C-terminus of surface proteins. Here we show that when placed into the middle of a polypeptide chain, the sorting signal causes the specific cleavage of the precursor as well as the cell wall anchoring of its N- terminal fragment, while the C-terminal fragment remains within the cytoplasm. N-terminal sequencing of the C-terminal cleavage fragment suggests that the cleavage site is located between threonine (T) and glycine (G) of the LPXTG motif, the signature sequence of cell wall sorting signals. All surface proteins harbouring an LPXTG sequence motif may therefore be cleaved and anchored by a universal mechanism. We also propose a novel hypothesis for the cell wall linkage of surface proteins in Gram-positive bacteria.

Notes: Summary The FeSII protein of Azotobacter vinelandii has been proposed to mediate the ‘conformational protection’ of the molybdenum-dependent nitrogenase components against oxygen inactivation. We have cloned and characterized the structural gene for the FeSII protein (the fesII Iocus). Hybridization studies did not reveal the presence of fesII-like genes in a number of diverse species of well-studied nitrogen-fixing bacteria, with the exception of Azotobacter chroococcum. The fesll locus is transcriptionally expressed during both nitrogen fixing and non-nitrogen fixing conditions, although the level of its message is up-regulated by approximately 2.5-fold during nitrogen fixation. The promoter region was identified by primer extension analysis, and is similar to other σ70-type promoters. Mutants devoid of the FeSII protein were constructed. These mutants possessed growth characteristics on a variety of carbon substrates during non-diazotrophic as well as diazotrophic growth that were essentially indistinguishable from the wild-type strain. Nevertheless, the nitrogenase activity in cell-free extracts is significantly more sensitive to irreversible oxygen inactivation in the mutants as compared with the wild type. When treated with 250 mM NaCI (a condition known to dissociate FeSII from nitrogenase components), the wild-type and mutant extracts were equally hypersensitive to oxygen Inactivation. Upon energy starvation, conditions in which ‘respiratory protection’ is inoperable, the MoFe and Fe proteins of nitrogenase are degraded much more rapidly in vivo in the deletion mutants, compared to the wild type. Strains relying on either the vanadium or the ‘iron-only’ alternative nitrogenases exhibited similar growth rates irrespective of the presence of absence of the FeSII protein, and the in vitro inactivation of the vanadium nitrogenase components was not affected by the lack of the FeSII protein. All in all, these results are consistent with a model whereby ‘respiratory protection’ is the major physiological mechanism responsible for the protection of all three nitrogenases during energy supplemented growth. Upon energy starvation, however, ‘conformational protection’ mediated by the FeSII protein is capable of temporarily protecting the conventional molybdenum nitrogenase components from inactivation and subsequent degradation.

Notes: Genes for biosynthesis of a Streptomyces sp. FR-008 heptaene macrolide antibiotic with antifungal and mosquito larvicidal activity were cloned in Escherichia coli using heterologous DNA probes. The cloned genes were implicated in heptaene biosynthiesis by gene replacement. The FR-008 antibiotic contains a 38-membered, poiyketide-derived macrolide ring. Southern hybridization using probes encoding domains of the type i modular erythromycin polyketide synthase (PKS) showed that the Streptomyces sp. FR-008 PKS gene cluster contains repeated sequences spanning c. 105 kb of contiguous DNA; assuming c. 5 kb for each PKS module, this is in striking agreement with the expectation for the 21-step condensation process required for synthesis of the FR-008 carbon chain. The methods developed for transformation and gene replacement in Streptomyces sp. FR-008 make it possible to genetically manipulate polyene macrolide production, and may later lead to the biosynthesis of novel polyene macrolides.

Notes: Interactions of Opc-expressing Neisseria meningitidis with polarized and non-polarized human umbilical vein endothelial cells (Huvecs) were investigated. Metabolic inhibitors and cytochalasin D treatment showed that host cellular and cytoskeletal functions were important for Opc-expressing bacterial association with Huvecs at the apical surface. In addition, this interaction required the presence of serum in the incubation medium whilst association with nonpolarized cells did not require serum. Pre-exposure of Opc-expressing bacteria to serum was sufficient to increase the number of bacterial interactions at the apical surface; B306, a monoclonal antibody (mAb) against Opc, inhibited these interactions, suggesting that Ope binds to serum factor(s) and this in turn increases adherence to Huvecs. The receptors involved in this ‘sandwich’ adherence belong to the integrin family since the interaction was inhibited by peptides containing the amino acid sequence arginine-glycine-aspartic acid (RGD) and the tetrapeptide RGDS (but not the peptide RGES) was inhibitory. Non-polarized cells appeared to expose receptors/sites that bound to Opc-expressing bacteria directly, did not require serum factors and were not inhibited by RGD-containing peptides. Serum-dependent interactions of Opc-expressing bacteria to apical surface was inhibited significantly by severai mAbs against avβ3 integrins. Some mAbs against α5 and β1 caused partial inhibition; antibodies that did not block the function of β1 integrins or the mAbs against α2 integrins were not inhibitory to bacterial interactions with Huvecs. Purified vitronectin supported adherence of Opc-expressing bacteria to Huvecs but not of Opc-bacteria. These interactions were inhibited by mAb B306 against Opc, by RGDS peptides as well as by blocking antibodies directed against αvβ-3 but not antibodies against other integrins. These data suggest that a sequence of molecular events resulting in trimolecular complexes at the endothelial surface may drive neisserial invasion of Huvesc. The expression of Opc appears to enable bacteria to utilize the normal signal-transduction mechanism of host cells via ligands in sera that adhere to endothelial cell integrins.

Notes: The toxicity to mosquito larvae of the parasporal body produced by Bacillus thuringiensis subsp. israelensis and the PG-14 isolate of B. thuringiensis subsp. morrisoni is at least 20-fold greater than any of the four mosquitocidal proteins of which It is composed (CytA, CrylVA, B, and D). This high toxicity is postulated to be due to synergistic interactions among parasporal proteins. However, this remains controversial because values reported for the specific toxicity of individual proteins, especially the CytA protein, vary widely owing to the methods used to purify and assay toxins against larvae. In an attempt to resolve questions of purity, specific toxicity, and synergism, individual genes encoding the CytA and CrylVD toxins were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis cells using the shuttle vector pHT3101. CytA and CryIVD inclusions were purified and their toxicity was determined alone and when combined at different ratios using bio-assays against first instars of Aedes aegypti. The LC50 for the CytA inclusion was 60 ng ml−1, whereas the LC50 for the CryIVD was 85ng ml−1 In comparison, the LC50s for different combinations of CytA and CrylVD inclusions ranged from 12–15 ng ml−1, 4–5 times higher than the toxicity of either protein alone, demonstrating marked synergism between these two proteins. These results suggest that the high toxicity of the wild-type parasporal bodies of B. thuringiensis subspp. israelensis and morrisoni Is due to synergism among three or four of their major proteins.

Notes: Threonine is found at the third position of the second α-helix in the helix-turn-helix motifs of most bacterial DNA-binding proteins. To investigate the role of this conserved residue in Escherichia coli Trp repressor function, plasmids encoding mutant Trp repressers with each of the 19 amino acid changes of Thr-81 were made by site-directed mutagenesis. All 19 changes decrease the activity of Trp holorepressor, indicating that the Thr-81 side-chain is critical for TrpR function. Three mutant repressors, Ser-81, Lys-81 and Arg-81, retain partial DNA-binding activity and inhibit transcription from the wild-type trp promoter/operator complex; challenge-phage assays show that Ser-81 and Lys-81 holorepressors have altered DNA-binding specificities. The side-chain of Thr-81 may make direct contacts with base pairs 4 and 3 of the trp operator, consistent with the nuclear magnetic resonance solution structures of the holorepressoroperator complex.

Notes: Expression from both the Escherichia coli nir and nrf promoters is dependent on anaerobic induction by FNR but is further regulated by NarL and NarP in response to the presence of nitrite and nitrate in the growth medium. The nir promoter is activated by NarL in response to nitrate and nitrite and activated by NarP in response to nitrate but not nitrite. The effects of point mutations suggest that NarL and NarP both bind to the same target, which is a pair of heptamer sequences organized as an inverted repeat, centred 691/2 bp upstream of the transcript startpoint. The nrf promoter can be activated by either NarP or NarL in response to nitrite but is repressed by NarL in response to nitrate. Mutational analysis of the nrf promoter has been exploited to corroborate the location of the -10 hexamer and the FNR-binding site, and to find the sites essential for nitrite-dependent activation and nitrate-dependent repression. Optimal activation by NarP or NarL in response to nitrite requires an inverted pair of heptamer sequences, similar to that found at the nir promoter, but centred 741/2 bp upstream from the transcript start. NarL-dependent repression by nitrate is due to two heptamer sequences that flank the FNR-binding sequence. We conclude that NarL and NarP bind to the same heptamer sequences, but that the affinities for the two factors vary from site to site.

Notes: Type 4 fimbriae (or pili) are associated with a form of bacterial surface translocation known as twitching motility. Fimbriae are also associated with sensitivity to certain bacteriophages such as PO4. Transposon mutagenesis was used to generate a library of Pseudomonas aeruginosa mutants which lack the spreading-colony morphology characteristic of twitching motility. in four of these mutants the transposon was found to be located in the vicinity of the previously described pilT locus, but in only one case was it found to have inserted within the pilT coding sequence. Two twitching-motility mutants originally isolated by Bradley, K2.2, and PAO2001.2, which have been widely used in studies of P. aeruginosa fimbrial structure and expression, were also shown to affect pilT and to comprise a small deletion and a frameshift mutation, respectively. The other three transposon mutations were found to have occurred within a new gene located directly downstream of pilT. This gene, termed pilU, encodes a 382-amino-acid protein closely related to PilT and to other members of a family of putative nucleotide-binding proteins which are involved in the assembly of ceil surface-associated complexes. Furthermore, the pilT and pilU genes appear to be independently expressed. Like pilT mutants, the pilU mutants were hyperfimbriate, but in neither case was this associated with an increase in transcription of the fimbrial subunit gene pilA. However, in contrast to pilT mutants, the pilU mutants had not also acquired resistance to infection by bacteriophage P04. A broader survey showed differential patterns of sensitivity to various fimbrial-specific phages among the pilU mutants and other twitching-motility mutants in the transposon library. The fact that twitching motility is not obligatorily associated with phage sensitivity suggests that the latter may not be directly dependent upon fimbrial function but rather may be a consequence of some common factor(s) involved in their assembly or export pathways.

Notes: We have used the toxic non-metabolizabie glucose/ mannose analogue 2-deoxygiucose to isolate a comprehensive collection of mutants of the phosphoenoipyruvate:sugar phosphotransferase system from Streptococcus salivarius. To increase the range of possible mutations, we isolated spontaneous mutants on different media containing 2-deoxyglucose and various metabolizable sugars, either lactose, meli-biose, galactose or fructose. We found that the frequency at which 2-deoxygiucose-resistant mutants Were isolated varied according to the growth substrate. The highest frequency was obtained with the combination galactose and 2-deoxygiucose and was 15-fold higher than the rate observed with the mixture melibiose and 2-deoxygiucose, the combination that gave the lowest frequency. By combining results from: (i) Western biol analysis of IIIMan, a specific component of the phosphoenolpyruvate:mannose phosphotransferase system in S. salivarius; (ii) rocket immunoelectrophoresis of HPr and EI, the two general energy-coupling proteins of the phosphotransferase system; and (iii) from gene sequencing, mutants could be assigned to seven classes. Class 1 was composed of strains devoid of IIIManL, a low-molecular-weight form of IIIManL (35200), class 2 was composed of strains exhibiting a reduced level of IIIManL, class 3 was composed of strains devoid of both forms of IIIMan (IIIManL as well as IIIManH, the high-molecular-weight form of IIIMan (38900)), class 4 was composed of mutants bearing a mutation in ptsH, the gene encoding HPr, class 5 was composed of mutants bearing a mutation in ptsl, the gene encoding EI, class 6 was composed of 2-deoxygiucose-resistant strains without any apparent defect in PTS components, and class 7 was composed of strains possessing both forms of IIIMan but abnormal levels of HPr and/or EI without any mutation in the ptsH and/or the ptsI genes. Preliminary characterization of representative strains of each class is reported.

Notes: Colicin A is a pore-forming bacteriocin that depends upon the Tol proteins in order to be transported from its receptor at the outer membrane surface to its target, the inner membrane. The presequence of yeast mitochondria cytochrome c1 (pc1) as well as the first 167 amino acids of cytochrome b2 (pb2) were fused to the pore-forming domain of colicin A (pfColA). Both hybrid proteins (pc1-pfColA and pb2-pfColA) were cytotoxic for Escherichia coli strains devoid of colicin A immunity protein whereas the pore-forming domain without presequence had no lethal effect. The entire precursors and their processed forms were found entirely associated with the bacterial inner membrane and their cytotoxicities were related to their pore-forming activities. The proteins were also shown to kill the tol bacterial strains, which are unable to transport colicins. In addition, we showed that both the cytochrome C1 presequence fused to the dihydrofolate reductase (pc1-DHFR) and the cytochrome c, presequence moiety of pc1-pfColA were translocated across inverted membrane vesicles. Our results indicated that: (i) pc1-pfColA produced in the cell cytoplasm was able to assemble in the inner membrane by a mechanism independent of the tol genes; (ii) the inserted pore-forming domain had a channel activity; and (ii) this channel activity was inhibited within the membrane by the immunity protein.

Notes: The sequence encompassing the cai genes of Escherichia coli, which encode the carnitine pathway, has been determined. Apart from the already identified caiB gene coding for the carnitine dehydratase, five additional open reading frames were identified. They belong to the caiTABCDE operon, which was shown to be located at the first minute on the chromosome and transcribed during anaerobic growth in the presence of carnitine. The activity of carnitine dehydratase was dependent on the CRP regulatory protein and strongly enhanced in the absence of a functional H-NS protein, in relation to the consensus sequences detected in the promoter region of the cai operon. In vivo expression studies led to the synthesis of five polypeptides in addition to CaiB, with predicted molecular masses of 56 613 Da (CaiT), 42 564 Da (CaiA), 59311 Da (CaiC), 32 329 Da (CaiD) and 21 930 Da (CaiE). Amino acid sequence similarity or enzymatic analysis supported the function assigned to each protein. CaiT was suggested to be the transport system for carnitine or betaines, CaiA an oxidoreduction enzyme, and CaiC a crotonobetaine/carnitine CoA ligase. CaiD bears strong homology with enoyl hydratases/isomerases. Overproduction of CaiE was shown to stimulate the carnitine racemase activity of the CaiD protein and to markedly increase the basal level of carnitine dehydratase activity. It is inferred that CaiE is an enzyme involved in the synthesis or the activation of the still unknown cofactor required for carnitine dehydratase and carnitine racemase activities. Taken together, these data suggest that the carnitine pathway in E. coli resembles that found in a strain situated between Agrobacterium and Rhizobium.

Notes: The chb1 gene, which encodes the unique lectin-like α-chitin-binding protein CHB1 of Streptomyces olivaceoviridis, was cloned. Transformants of Streptomyces lividans harbouring the plasmid pCHB10 overproduced the extracellular CHB1 protein; the protein showed neither enzymatic nor antifungal activity. Biochemical analyses and immunofluorescence microscopy indicated that CHB1 binds strongly to α-chitin, but neither to chitosan and β-chitin, nor to various types of cellulose. Within hyphae of fungi, the relative location of crystalline chitin was visualized with fluor-escein-labelled CHB1. These studies suggest that the new protein could serve as a tool to identify α-chitin within different organisms. The chb1 gene consists of a reading frame of 603 bp and its transcription occurred only if the Streptomyces strain was cultivated with chitin as the sole carbon source. The deduced mature CHB1 protein (18.7 kDa) shows no apparent similarity to any known protein. Within a region containing 100 residues of the deduced CHB1 protein, four tryptophan and two asparagine residues as well as one glycine and one cysteine residue were identified, the relative positions of which are analogous to those of several cellulose-binding domains of bacterial glycohydrolases. The results of spectroscopical studies suggest a possible involvement of tryptophan residues in the interaction of CHB1 with α-chitin.

Notes: Mycoplasmas have originated from Gram-positive bacteria via rapid degenerative evolution. The results of previous investigations of mycoplasmal DNA polymerases suggest that the process of evolution has wrought a major simplification of the typical Gram-positive bacterial DNA polymerase profile, reducing it from three exonuclease (exo)-positive enzymes to a single exo-negative species. The objective of this work was to rigorousiy investigate this suggestion, focusing on the evolutionary fate of DNA polymerase III (Pol III), the enzyme which Gram-positive bacteria specifically require for replicative DNA synthesis. The approach used Mycoplasma pulmonis as the model organism and exploited structural gene cloning, enzymology, and Pol III-specific inhibitors of the HPUra class as investigative tools. Our results indicate that M. pulmonis has strongly conserved a single copy of a structural gene homologous to polC, the Gram-positive bacterial gene encoding Pol III M. pulmonis was found to possess a DNA polymerase that displays the size, primary structure, exonuclease activity, and level of HPUra sensitivity expected of a prototypical Gram-positive Pol III. The high level of sensitivity of M. pulmonis growth to Gram-positive Pol III-selective inhibitors of the HPUra type strongly suggests that Mycoplasma has conserved not only the basic structure of Pol III, but also its essential replicative function. Evidence for a second, HPUra-resistant polymerase activity in M. pulmonis is also described, indicating that the DNA polymerase composition of Mycoplasma is complex and closer to that of Gram-positive bacteria than previously thought.