Bixing Huang

FimX, a Multidomain Protein Connecting Environmental Signals to Twitching Motility in Pseudomonas aeruginosa

Twitching motility is a form of surface translocation mediated by the extension, tethering, and retraction of type IV pili. Three independent Tn5-B21 mutations of Pseudomonas aeruginosa with reduced twitching motility were identified in a new locus which encodes a predicted protein of unknown function annotated PA4959 in the P. aeruginosa genome sequence. Complementation of these mutants with the wild-type PA4959 gene, which we designated fimX, restored normal twitching motility. fimX mutants were found to express normal levels of pilin and remained sensitive to pilus-specific bacteriophages, but they exhibited very low levels of surface pili, suggesting that normal pilus function was impaired. The fimX gene product has a molecular weight of 76,000 and contains four predicted domains that are commonly found in signal transduction proteins: a putative response regulator (CheY-like) domain, a PAS-PAC domain (commonly involved in environmental sensing), and DUF1 (or GGDEF) and DUF2 (or EAL) domains, which are thought to be involved in cyclic di-GMP metabolism. Red fluorescent protein fusion experiments showed that FimX is located at one pole of the cell via sequences adjacent to its CheY-like domain. Twitching motility in fimX mutants was found to respond relatively normally to a range of environmental factors but could not be stimulated by tryptone and mucin. These data suggest that fimX is involved in the regulation of twitching motility in response to environmental cues.

Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa

Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for colonization of host tissues and are associated with a form of surface translocation termed twitching motility. Twitching motility in P. aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK. Here we describe another three components of this pathway: ChpA, ChpB and ChpC, as well as two downstream genes, ChpD and ChpE, which may also be involved. The central component of the pathway, ChpA, possesses nine potential sites of phosphorylation: six histidine‐containing phosphotransfer (HPt) domains, two novel serine‐ and threonine‐containing phosphotransfer (SPt, TPt) domains and a CheY‐like receiver domain at its C‐terminus, and as such represents one of the most complex signalling proteins yet described in nature. We show that the Chp chemosensory system controls twitching motility and type IV pili biogenesis through control of pili assembly and/or retraction as well as expression of the pilin subunit gene pilA. The Chp system is also required for full virulence in a mouse model of acute pneumonia.

Prediction of protein accessible surface areas by support vector regression.

A novel support vector regression (SVR) approach is proposed to predict protein accessible surface areas (ASAs) from their primary structures. In this work, we predict the real values of ASA in squared angstroms for residues instead of relative solvent accessibility. Based on protein residues, the mean and median absolute errors are 26.0 Å2 and 18.87 Å2, respectively. The correlation coefficient between the predicted and observed ASAs is 0.66. Cysteine is the best predicted amino acid (mean absolute error is 13.8 Å2 and median absolute error is 8.37 Å2), while arginine is the least predicted amino acid (mean absolute error is 42.7 Å2 and median absolute error is 36.31 Å2). Our work suggests that the SVR approach can be directly applied to the ASA prediction where data preclassification has been used. Proteins 2004.

An interactive web-based Pseudomonas aeruginosa genome database: discovery of new genes, pathways and structures

Using the complete genome sequence of Pseudomonas: aeruginosa PAO1, sequenced by the Pseudomonas: Genome Project (ftp://ftp.pseudomonas. com/data/pacontigs.121599), a genome database (http://pseudomonas. bit.uq.edu.au/) has been developed containing information on more than 95% of all ORFs in Pseudomonas: aeruginosa. The database is searchable by a variety of means, including gene name, position, keyword, sequence similarity and Pfam domain. Automated and manual annotation, nucleotide and peptide sequences, Pfam and SMART domains (where available), Medline and GenBank links and a scrollable, graphical representation of the surrounding genomic landscape are available for each ORF. Using the database has revealed, among other things, that P. aeruginosa contains four chemotaxis systems, two novel general secretion pathways, at least three loci encoding F17-like thin fimbriae, six novel filamentous haemagglutinin-like genes, a number of unusual composite genetic loci related to vgr/RHS: elements in Escherichia coli, a number of fix-like genes encoding a micro-oxic respiration system, novel biosynthetic pathways and 38 genes containing domains of unknown function (DUF1/DUF2). It is anticipated that this database will be a useful bioinformatic tool for the Pseudomonas: community that will continue to evolve.

Comparison of multiplex PCR with conventional biochemical methods for the identification of Listeria spp. isolates from food and clinical samples in Queensland, Australia.

Listeria monocytogenes is an important foodborne pathogen with high mortality. L. monocytogenes and five other Listeria species can frequently be found in the same sample. To identify Listeria isolates found in foods to the species level, two multiplex PCRs were designed. The PCR and conventional biochemical methods were compared for the identification of 456 Listeria isolates collected from routine food quality monitoring schemes between June 2004 and February 2006 and for 62 L. monocytogenes isolates from patients between 1999 and 2005. The results showed that the PCR and biochemical methods had 100% agreement in Listeria identification. The distribution of Listeria species from foods was as follows: L. monocytogenes, 50.4%; L. innocua, 33.8%; L. welshimeri, 14.9%; L. seeligeri, 0.7%; L. grayi, 0.2%; and L. ivanovii, 0.0%. Additional analyses were performed to identify the major serotypes (1/2a, 1/2b, 1/2c, and 4b) and the three lineages of L. monocytogenes isolates from foods and patients, with 1/2a (69.6%) and 1/2b (21.7%) dominating the food isolates and 1/2b (54.8%) and 4b (30.7%) dominating the patient isolates. The lineage results showed that isolates of 1/2a and 1/2c belonged to lineage II and that isolates of 1/2b and 4b belonged to lineage I. The multiplex PCRs for Listeria identification that have been established provide an accurate and rapid method for food quality control. This study has provided the basic knowledge of distribution of Listeria species and L. monocytogenes serotypes in Queensland, Australia, which is useful for epidemiological investigations of listeriosis.

A Survey of the Bacteriological Quality of Preroasted Peanut, Almond, Cashew, Hazelnut, and Brazil Nut Kernels Received into Three Australian Nut-Processing Facilities over a Period of 3 Years

There is little information about bacteriological quality of preroasted kernels available in the public domain. An investigation of the bacteriological quality of preroasted peanut, almond, cashew, hazelnut, and Brazil nut kernels received into three Australian nut-processing facilities was performed over a period of 3 years. A total of 836 samples were analyzed for aerobic plate count, and 921 samples for Salmonella and Escherichia coli. The 921 samples included 653 peanut, 100 cashew, 60 almond, 60 Brazil nut, and 48 hazelnut kernels. There was no E. coli detected in any sample. Salmonella subsp. II (Fremantle) was detected in one raw almond sample. The aerobic plate count percentages of positive samples with counts above the detection level of the plating method used (100 CFU/g) for peanuts, almonds, cashews, hazelnuts, and Brazil nuts were 84, 78, 74, 50, and 45%, respectively. Of the samples containing more than this detection limit, the means were 4.5, 4.4, 3.1, 2.5, and 3.8 log CFU/g respectively. Although roasted kernel quality was not within the scope of this survey, raw microbial bioload would be expected to reduce on roasting. The bacteriological quality of preroasted peanut, almond, cashew, hazelnut, and Brazil nut kernels received into nut-processing facilities in Australia does not appear to suggest a public health concern.

tonB3 Is Required for Normal Twitching Motility and Extracellular Assembly of Type IV Pili

Three mutants with Tn5-B21 insertion in tonB3 (PA0406) of Pseudomonas aeruginosa exhibited defective twitching motility and reduced assembly of extracellular pili. These defects could be complemented with wild-type tonB3.

A Predominant and Virulent Legionella pneumophila Serogroup 1 Strain Detected in Isolates from Patients and Water in Queensland, Australia, by an Amplified Fragment Length Polymorphism Protocol and Virulence Gene-Based PCR Assays

ABSTRACT In epidemiological investigations of community legionellosis outbreaks, knowledge of the prevalence, distribution, and clinical significance (virulence) of environmental Legionella isolates is crucial for interpretation of the molecular subtyping results. To obtain such information for Legionella pneumophila serogroup 1 isolates, we used the standardized amplified fragment length polymorphism (AFLP) protocol of the European Working Group on Legionella Infection to subtype L. pneumophila SG1 isolates obtained from patients and water sources in Queensland, Australia. An AFLP genotype, termed AF1, was predominant in isolates from both patients (40.5%) and water (49.0%). The second most common AFLP genotype found in water isolates was AF16 (36.5%), but this genotype was not identified in the patient isolates. When virulence gene-based PCR assays for lvh and rtxA genes were applied to the isolates from patients and water, nearly all (65 of 66) AF1 strains had both virulence genes, lvh and rtxA. In contrast, neither the lvh nor the rtxA gene was found in the AF16 strains, except for one isolate with the rtxA gene. It appears that this may explain the failure to find this genotype in the isolates from patients even though it may be common in the environment. In view of the evidence that the AF1 genotype is the most common genotype among strains found in patients and water sources in this region, any suggested epidemiological link derived from comparing the AF1 genotype from patient isolates with the AF1 genotype from environmental isolates must be interpreted and acted on with caution. The use of virulence gene-based PCR assays applied to environmental samples may be helpful in determining the infection potential of the isolates involved.

Observation of a New Pattern in Serogroup-Related PCR Typing of Listeria monocytogenes 4b Isolates

ABSTRACT Molecular serogroup-related PCR typing has made the determination of serotypes of Listeria monocytogenes isolates easy and rapid. Amplification of selected lineage- and serotype-related genes can produce serotype patterns reflecting the four major serotypes, 1/2a, 1/2b, 1/2c, and 4b. We found that four isolates in our routine testing had a pattern with the four bands lmo0737, ORF2110, ORF2819, and prs positive, a pattern which has not been previously reported in the literature. After testing with a lineage-specific PCR, hybridization, and conventional agglutination serotyping, the isolates with the new pattern were considered to be serotype 4b.

Bacteriological profile of raw, frozen chicken nuggets.

The bacteriological profile of raw, frozen chicken nuggets manufactured at a chicken processing facility in Queensland, Australia, was determined. Chicken nuggets are manufactured by grinding poultry, adding premixes to incorporate spices, forming the meat to the desired size and shape, applying a batter and breading, freezing, and packaging. A total of 300 frozen batches were analyzed for aerobic plate count, Escherichia coli, and Salmonella over a period of 4 years. The mean of the aerobic plate count was 5.4 log CFU/g, and counts at the 90th, 95th, and 99th percentiles were 5.7, 5.9, and 6.5 log CFU/g, respectively. The maximum number of bacteria detected was 6.6 log CFU/g. E. coli prevalence was 47%, and of the positive samples, the mean was 1.9 log CFU/g; counts at the 90th, 95th, and 99th percentiles were 2.3, 2.4, and 2.8 log CFU/g, respectively. The maximum number of E. coli was 2.9 log CFU/g. The Salmonella prevalence was 8.7%, and 57.7% of these isolates were typed as Salmonella subspecies II 4,12,[27]:b:[e,n,x] (Sofia), a low-virulence serotype well adapted to Australian poultry flocks. There was a significant relationship (P < 0.05) between season and both aerobic plate counts and E. coli counts, and no correlation between E. coli counts and Salmonella prevalence. This study provides valuable data on the bacteriological quality of raw, frozen chicken nuggets.