Theoretical insights into bacterial chemotaxis

Research into understanding bacterial chemotactic systems has become a paradigm for Systems Biology. Experimental and theoretical researchers have worked hand-in-hand for over 40 years to understand the intricate behavior driving bacterial species, in particular how such small creatures, usually not more than 5 µm in length, detect and respond to small changes in their extracellular environment. In this review we highlight the importance that theoretical modeling has played in providing new insight and understanding into bacterial chemotaxis. We begin with an overview of the bacterial chemotaxis sensory response, before reviewing the role of theoretical modeling in understanding elements of the system on the single cell scale and features underpinning multiscale extensions to population models. WIREs Syst Biol Med 2012 doi: 10.1002/wsbm.1168 For further resources related to this article, please visit the WIREs website.

Intestinal injury and endotoxemia in children undergoing surgery for congenital heart disease

RATIONALE: Children with congenital heart disease are at risk of gut barrier dysfunction and translocation of gut bacterial antigens into the bloodstream. This may contribute to inflammatory activation and organ dysfunction postoperatively. OBJECTIVES: To investigate the role of intestinal injury and endotoxemia in the pathogenesis of organ dysfunction after surgery for congenital heart disease. METHODS: We analyzed blood levels of intestinal fatty acid binding protein and endotoxin (endotoxin activity assay) alongside global transcriptomic profiling and assays of monocyte endotoxin receptor expression in children undergoing surgery for congenital heart disease. MEASUREMENTS AND MAIN RESULTS: Levels of intestinal fatty acid binding protein and endotoxin were greater in children with duct-dependent cardiac lesions. Endotoxemia was associated with severity of vital organ dysfunction and intensive care stay. We identified activation of pathogen-sensing, antigen-processing, and immune-suppressing pathways at the genomic level postoperatively and down-regulation of pathogen-sensing receptors on circulating immune cells. CONCLUSIONS: Children undergoing surgery for congenital heart disease are at increased risk of intestinal mucosal injury and endotoxemia. Endotoxin activity correlates with a number of outcome variables in this population, and may be used to guide the use of gut-protective strategies.

A process for analysis of microarray comparative genomics hybridisation studies for bacterial genomes

Background: Microarray based comparative genomic hybridisation (CGH) experiments have been used to study numerous biological problems including understanding genome plasticity in pathogenic bacteria. Typically such experiments produce large data sets that are difficult for biologists to handle. Although there are some programmes available for interpretation of bacterial transcriptomics data and CGH microarray data for looking at genetic stability in oncogenes, there are none specifically to understand the mosaic nature of bacterial genomes. Consequently a bottle neck still persists in accurate processing and mathematical analysis of these data. To address this shortfall we have produced a simple and robust CGH microarray data analysis process that may be automated in the future to understand bacterial genomic diversity. Results: The process involves five steps: cleaning, normalisation, estimating gene presence and absence or divergence, validation, and analysis of data from test against three reference strains simultaneously. Each stage of the process is described and we have compared a number of methods available for characterising bacterial genomic diversity, for calculating the cut-off between gene presence and absence or divergence, and shown that a simple dynamic approach using a kernel density estimator performed better than both established, as well as a more sophisticated mixture modelling technique. We have also shown that current methods commonly used for CGH microarray analysis in tumour and cancer cell lines are not appropriate for analysing our data. Conclusion: After carrying out the analysis and validation for three sequenced Escherichia coli strains, CGH microarray data from 19 E. coli O157 pathogenic test strains were used to demonstrate the benefits of applying this simple and robust process to CGH microarray studies using bacterial genomes.

A modified rapid enzymatic microtiter plate assay for the quantification of intracellular γ-aminobutyric acid and succinate semialdehyde in bacterial cells

The GABase assay is widely used to rapidly and accurately quantify levels of extracellular γ-aminobutyric acid (GABA). Here we demonstrate a modification of this assay that enables quantification of intracellular GABA in bacterial cells. Cells are lysed by boiling and ethanolamine-O-sulphate, a GABA transaminase inhibitor is used to distinguish between GABA and succinate semialdehyde.

Role of glutamate metabolism in bacterial responses towards acid and other stresses

Glutamate plays a central role in a wide range of metabolic processes in bacterial cells. This review focuses on the involvement of glutamate in bacterial stress responses. In particular it reviews the role of glutamate metabolism in response against acid stress and other stresses. The glutamate decarboxylase (GAD) system has been implicated in acid tolerance in several bacterial genera. This system facilitates intracellular pH homeostasis by consuming protons in a decarboxylation reaction that produces γ-aminobutyrate (GABA) from glutamate. An antiporter system is usually present to couple the uptake of glutamate to the efflux of GABA. Recent insights into the functioning of this system will be discussed. Finally the intracellular fate of GABA will also be discussed. Many bacteria are capable of metabolising GABA to succinate via the GABA shunt pathway. The role and regulation of this pathway will be addressed in the review. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Nanoscale zerovalent iron alters soil bacterial community structure and inhibits chloroaromatic biodegradation potential in Aroclor 1242-contaminated soil

Nanoscale zerovalent iron (nZVI) has potential for the remediation of organochlorine-contaminated environments. Environmental safety concerns associated with in situ deployment of nZVI include potential negative impacts on indigenous microbes whose biodegradative functions could contribute to contaminant remediation. With respect to a two-step polychlorinated biphenyl remediation scenario comprising nZVI dechlorination followed by aerobic biodegradation, we examined the effect of polyacrylic acid (PAA)-coated nZVI (mean diameter = 12.5 nm) applied at 10 g nZVI kg−1 to Aroclor-1242 contaminated and uncontaminated soil over 28 days. nZVI had a limited effect on Aroclor congener profiles, but, either directly or indirectly via changes to soil physico-chemical conditions (pH, Eh), nZVI addition caused perturbation to soil bacterial community composition, and reduced the activity of chloroaromatic mineralizing microorganisms. We conclude that nZVI addition has the potential to inhibit microbial functions that could be important for PCB remediation strategies combining nZVI treatment and biodegradation.

Studies into the role of the SEF14 fimbrial antigen in the pathogenesis of Salmonella enteritidis

To investigate the role of the SEF14 fimbrial antigen in pathogenesis, a single defined sefA (SEF14(-)) inactivated mutant of Salmonella enteritidis strain LA5 was constructed and tested in a number of biological assay systems. There was no significant difference between the wild-type strain and the isogenic SEF14(-) mutant in their abilities to adhere to and invade HEp-2 epithelial cells or their survival in mouse peritoneal macrophages, whereas the SEF14(-) mutant was ingested more rapidly by isolated human PMN. Both the strains colonized the intestine, invaded and spread systemically in 1 day-old chicks, laying hens and BALB/c mice equally well. A significantly greater number of chicks excreted the wildtype SEF14(+) strain during the first week following infection as compared to those infected with the SEF14(-) mutant. However, similar numbers of chicks excreted the two strains between 2 and 7 weeks after infection. These results indicate that possession of SEF14 fimbriae alone do not appear to play a significant role in the pathogenesis of S. enteritidis although its contribution to virulence may be dependent on the host species infected. (C) 1996 Academic Press Limited

Distribution, gene sequence and expression in vivo of the plasmid encoded fimbrial antigen of Salmonella serotype Enteritidis

The pefA gene which encoded the serotype associated plasmid (SAP) mediated fimbrial major subunit antigen of Salmonella enterica serotype Typhimurium shared genetic identity with 128 of 706 salmonella isolates as demonstrated by dot (colony) hybridization. Seventy-seven of 113 isolates of Typhimurium and individual isolates of serotypes Bovis-morbificans, Cholerae-suis and Enteritidis phage type 9b hybridized pefA strongly, whereas 48 isolates of Enteritidis hybridized pefA weakly and one Enteritidis isolate of phage type 14b failed to hybridize. Individual isolates of 294 serotypes and 247 individual isolates of serotype Dublin did not hybridize pefA. Southern hybridization of plasmids extracted from Enteritidis demonstrated that the pefA gene probe hybridized strongly an atypical SAP of 80 kb in size harboured by one Enteritidis isolate of phage-type 9b, whereas the typical SAP of 58 kb in size harboured by 48 Enteritidis isolates hybridized weakly. One Enteritidis isolate of phage type 14b which failed to hybridize pefA in dot (colony) hybridization experiments was demonstrated to be plasmid free. A cosmid library of Enteritidis phage type 4 expressed in Escherichia coli K12 was screened by hybridization for the presence of pef sequences. Recombinant clones which were deduced to harbour the entire pef operon elaborated a PEF-like fimbrial structure at the cell surface. The PEF-like fimbrial antigen was purified from one cosmid clone and used in western blot experiments with sera from chickens infected with Enteritidis phage-type 4. Seroconversion to the fimbrial antigen was observed which indicated that the Enteritidis PEF-like fimbrial structure was expressed at some stage during infection. Nucleotide sequence analysis demonstrated that the pefA alleles of Typhimurium and Enteritidis phage-type 4 shared 76% DNA nucleotide and 82% deduced amino acid sequence identity.

Spirochaetes and other bacterial species associated with bovine digital dermatitis

The 16S rRNA genes from spirochaetes associated with digital dermatitis of British cattle were amplified by polymerase chain reaction from digital dermatitis lesion biopsies using one universal and one treponeme-specific primer. Two treponemal sequences were identified both of which shared a high degree of homology with the oral pathogen Treponema denticola (98%). Two further 16S rRNA gene sequences were obtained and shared similarity to Bacteroides levii (99%) and Mycoplasma hyopharyngis (98%). Polymerase chain reaction with T. denticola-specific primers amplified a potential virulence gene from digital dermatitis lesions which shared a high degree of homology to the 46-kDa haemolysin gene of T. denticola. The significance of the presence of organisms in digital dermatitis lesions of the bovine foot which are closely related to oral pathogens is discussed.

The role of SEF14 and SEF17 fimbriae in the adherence of Salmonella enterica serotype Enteritidis to inanimate surfaces

To gain an understanding of the role of fimbriae and flagella in the adherence of Salmonella enterica serotype Enteritidis to inanimate surfaces, the extent of adherence of viable wild-type strains to a polystyrene microtitration plate was determined by a crystal violet staining assay, Elaboration of surface antigens by adherent bacteria was assayed by fimbriae- and flagella-specific ELISAs, Wild-type Enteritidis strains adhered well at 37 degrees C and 25 degrees C when grown in microtitration wells in Colonisation Factor Antigen broth, but not in other media tested, At 37 degrees C, adherent bacteria elaborated copious quantities of SEF14 fimbrial antigen, whereas at 25 degrees C adherent bacteria elaborated copious quantities of SEF17 fimbrial antigen. Non-fimbriate and non-flagellate knock-out mutant strains were also assessed in the adherence assay. Mutant strains unable to elaborate SEF14 and SEF17 fimbriae adhered poorly at 37 degrees C and 25 degrees C, respectively, but adherence was not abolished. Non-motile mutant strains showed reduced adherence whilst type-1, PEF and LPF fimbriae appeared not to contribute to adherence in this assay. These data indicate that SEF17 and SEF14 fimbriae mediate bacterial cell aggregation on inanimate surfaces under appropriate growth conditions.

The SEF14 fimbrial antigen of Salmonella enterica serovar Enteritidis is encoded within a pathogenicity islet

The DNA sequence of the chromosomal gene cluster encoding the SEF14 fimbriae of Salmonella enterica serovar Enteritidis was determined. Five contiguous open reading frames, sefABCDE, were identified. The sefE gene shared significant homology with araC-like positive regulators. Serovar-associated virulence plasmid (SAP) genes orf7,8,9 and pefI were identified immediately adjacent to the sef operon. The pefI gene encoded a putative regulator of the Plasmid-encoded fimbrial antigen (PEF) expression. The entire sef-pef region, Ranked by two IS-like elements, was inserted adjacent to leuX that encoded a transfer RNA molecule. The organisation of this region was suggestive of a classic pathogenicity islet. Southern hybridisation confirmed two copies of the SAP derived orf7,8,9 and pefI region in S. Enteritidis, one in the chromosome and one on the SAP. Of other group D Salmonella, only S. Blegdam and S. Moscow harboured both chromosomal and plasmid copies of pefI-orf9 region although polymorphism was evident. Crown Copyright (C) 2001 Published by Elsevier Science B.V. All rights reserved.

The O-antigen of Salmonella enterica serotype Enteritidis PT4: a significant factor in gastrointestinal colonisation of young but not newly hatched chicks

The lipopolysaccharide of Salmonella and other Gram negative pathogenic species has been implicated as a major virulence determinant and in this study we report the role of LPS of S. Enteritidis in the colonisation and persistent gastrointestinal infection of young poultry. The gene encoding the unique O-antigen ligase, waaL, was mutated by insertional inactivation in a well characterised S. Enteritidis strain, S1400/94. The waaL mutant, designated PCP, produced rough colonies on agar medium, did not agglutinate O9 antiserum, did not produce an LPS ladder on silver stained gels and was serum sensitive. PCP and a nalidixic acid marked derivative of S1400/94 (S1400/94 Nal(r)) were used to orally challenge young chicks, separately and together in competitive index experiments. At post-mortem examination of 1-day-old chicks challenged S1400/94 Nal(r) and PCP separately there were no significant differences in the numbers of S1400/94 Nal(r) and PCP bacteria in tissues sampled on days 1, 2. and 5. By day 42 after challenge S1400/94 Nal(r) bacteria were recovered in significantly higher numbers than PCP from the caecal contents (P < 0.001). In competitive index studies in the 1-day-old chick PCP colonised, invaded and persisted in lower numbers than S1400/94 Nal(r). In 4-week-old chicks challenged separately, PCP bacteria were recovered from all tissues examined in significantly lower numbers than S1400/94 Nal(r). In competitive index experiments in 4-week-old chicks, PCP was not detected at any site and at any time point. Therefore, the O-antigen of S. Enteritidis plays art important role in poultry infections although this role is less important in the newly hatched chick. Crown Copyright (C) 2004 Published by Elsevier B.V. All rights reserved.

In vivo characterization of Lactobacillus johnsonii FI9785 for use as a defined competitive exclusion agent against bacterial pathogens in poultry

Aims: To test the efficacy of Lactobacillus johnsonii FI9785 in reducing the colonization and shedding of Salmonella enterica serotype Enteritidis, Escherichia coli O78:K80 and Clostridium perfringens in poultry. Methods and Results: Specific pathogen-free chicks (1 day old) were dosed with a single oral inoculum of 1 x 10(9) CFU. Lactobacillus johnsonii FI9785 and 24 h later were challenged in separate experiments with S. Enteritidis (S1400, nal(r)) and E. coli O78:K80 (EC34195, nal(r)). There were no significant effects against S. Enteritidis whereas colonization of the small intestine by E. coli O78:K80 was reduced significantly. Both S. Enteritidis and E. coli colonized the caeca and colon to levels equivalent to control birds and there was no reduction in shedding as assessed by a semi-quantitative cloacal swabbing technique. Specific pathogen-free chicks (20 day old) were dosed with a single oral inoculum of 1 x 10(9) CFU L. johnsonii FI9785 and 24 h later were challenged with C. perfringens. A single oral dose of L. johnsonii FI9785 was sufficient to suppress all aspects of colonization and persistence of C. perfringens. Conclusions: Lactobacillus johnsonii FI9785 may be given to poultry for use as a competitive exclusion agent to control C. perfringens. Significance and Impact of the Study: Lactobacillus johnsonii FI9785 may be a valuable tool to control the endemic disease of necrotic enteritis, thereby reducing economic losses associated with reduced use of antimicrobials in the poultry industry.

Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.

A maize bacterial artificial chromosome (BAC) library from the European flint inbred line F2

We report here the construction and characterisation of a BAC library from the maize flint inbred line F2, widely used in European maize breeding programs. The library contains 86,858 clones with an average insert size of approximately 90 kb, giving approximately 3.2-times genome coverage. High-efficiency BAC cloning was achieved through the use of a single size selection for the high-molecular-weight genomic DNA, and co-transformation of the ligation with yeast tRNA to optimise transformation efficiency. Characterisation of the library showed that less than 0.5% of the clones contained no inserts, while 5.52% of clones consisted of chloroplast DNA. The library was gridded onto 29 nylon filters in a double-spotted 8 × 8 array, and screened by hybridisation with a number of single-copy and gene-family probes. A 3-dimensional DNA pooling scheme was used to allow rapid PCR screening of the library based on primer pairs from simple sequence repeat (SSR) and expressed sequence tag (EST) markers. Positive clones were obtained in all hybridisation and PCR screens carried out so far. Six BAC clones, which hybridised to a portion of the cloned Rp1-D rust resistance gene, were further characterised and found to form contigs covering most of this complex resistance locus.