Molecular analysis of type 3 fimbrial genes from <Emphasis Type="Italic">Escherichia coli</Emphasis>, <Emphasis Type="Italic">Klebsiella</Emphasis> and <Emphasis Type="Italic">Citrobacter</Emphasis> species

Background  

Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii.     

<Emphasis Type="Italic">In vitro</Emphasis> activity of telithromycin against <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> at epithelial lining fluid concentrations

Background  

Haemophilus influenzae is one of the main aetiological agents of community-acquired respiratory tract infections. The primary aim of this study was to evaluate the antibacterial activity of telithromycin against H. influenzae clinical isolates showing different pattern of resistance in comparison with azithromycin and clarithromycin at 1/4 ×, 1/2 ×, 1 ×, 2 ×, 4 × minimum inhibitory concentration (MIC) and to peak concentrations in epithelial lining fluid (ELF). The secondary aim was to determine the influence of CO₂ enriched atmosphere on bacterial susceptibility.     

Prevalence of genetic differences in phosphorylcholine expression between nontypeable <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> and <Emphasis Type="Italic">Haemophilus haemolyticus</Emphasis>

Background  

Although non-typeable (NT) Haemophilus influenzae and Haemophilus haemolyticus are closely related human commensals, H. haemolyticus is non-pathogenic while NT H. influenzae is an important cause of respiratory tract infections. Phase-variable phosphorylcholine (ChoP) modification of lipooligosaccharide (LOS) is a NT H. influenzae virulence factor that, paradoxically, may also promote complement activation by binding C-reactive protein (CRP). CRP is known to bind more to ChoP positioned distally than proximally in LOS, and the position of ChoP within LOS is dictated by specific licD alleles (designated here as licD _I , licD _III , and licD _IV ) that are present in a lic1 locus. The lic1 locus contains the licA-licD genes, and ChoP-host interactions may also be influenced by a second lic1 locus that allows for dual ChoP substitutions in the same strain, or by the number of licA gene tetranucleotide repeats (5'-CAAT-3') that reflect phase-variation mutation rates.     

Mapping phosphoproteins in <Emphasis Type="Italic">Mycoplasma genitalium</Emphasis> and <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis>

Background  

Little is known regarding the extent or targets of phosphorylation in mycoplasmas, yet in many other bacterial species phosphorylation is known to play an important role in signaling and regulation of cellular processes. To determine the prevalence of phosphorylation in mycoplasmas, we examined the CHAPS-soluble protein fractions of Mycoplasma genitalium and Mycoplasma pneumoniae by two-dimensional gel electrophoresis (2-DE), using a combination of Pro-Q Diamond phosphoprotein stain and ³³P labeling. Protein spots that were positive for phosphorylation were identified by peptide mass fingerprinting using MALDI-TOF-TOF mass spectrometry.     

Common genomic features of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp. <Emphasis Type="Italic">doylei</Emphasis> strains distinguish them from <Emphasis Type="Italic">C. jejuni</Emphasis> subsp. <Emphasis Type="Italic">jejuni</Emphasis>

Background  

Campylobacter jejuni has been divided into two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Nearly all of the C. jejuni strains isolated are Cjj; nevertheless, although Cjd strains are isolated infrequently, they differ from Cjj in two key aspects: they are obtained primarily from human clinical samples and are associated often with bacteremia, in addition to gastroenteritis. In this study, we utilized multilocus sequence typing (MLST) and a DNA microarray-based comparative genomic indexing (CGI) approach to examine the genomic diversity and gene content of Cjd strains.     

Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Phospholipid biosynthesis commences with the acylation of glycerol-3-phosphate (G3P) to form 1-acyl-G3P. This step is catalyzed by the PlsB protein in Escherichia coli. The gene encoding this protein has not been identified, however, in the majority of bacterial genome sequences, including that of Bacillus subtilis. Recently, a new two-step pathway catalyzed by PlsX and PlsY proteins for the initiation of phospholipid formation in Streptococcus pneumoniae has been reported.     

A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of <Emphasis Type="Italic">Entamoeba histolytica</Emphasis>, <Emphasis Type="Italic">E. moshkovskii</Emphasis> and <Emphasis Type="Italic">E. dispar</Emphasis> DNA in stool samples

Background  

E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples.     

Molecular Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis>

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.     

A <Emphasis Type="Italic">gyrB</Emphasis>-targeted PCR for Rapid Identification of <Emphasis Type="Italic">Salmonella</Emphasis>

Salmonella causes the majority of infections in humans and homeothermic animals. This article describes a specific polymerase chain reaction (PCR) method developed for a rapid identification of Salmonella. A gyrB-targeted species-specific primer pair, S-P-for (5′-GGT GGT TTC CGT AAA AGT A-3′) and S-P-rev (5′-GAA TCG CCT GGT TCT TGC-3′), was successfully designed. PCR with all the Salmonella strains produced a 366- bp DNA fragment that was absent from all the non-Salmonella strains tested. The detection limit of the PCR was 0.01 ng with genomic DNA or 3.2 cells per assay. Good specificity was also demonstrated by fecal samples, from which only the gyrB gene of Salmonella was amplified. Using the culture-PCR method, 27 isolates on Salmonella-Shigella (SS) medium were rapidly identified as Salmonella, which was confirmed by the sequencing of the gyrB gene.     

Identification and characterization of a nontypeable <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> putative toxin-antitoxin locus

Background  

Certain strains of an obligate parasite of the human upper respiratory tract, nontypeable Haemophilus influenzae (NTHi), can cause invasive diseases such as septicemia and meningitis, as well as chronic mucosal infections such as otitis media. To do this, the organism must invade and survive within both epithelial and endothelial cells. We have identified a facilitator of NTHi survival inside human cells, virulence-associated protein D (vapD _Hi , encoded by gene HI0450). Both vapD _Hi and a flanking gene, HI0451, exhibit the genetic and physical characteristics of a toxin/antitoxin (TA) locus, with VapD _Hi serving as the toxin moiety and HI0451 as the antitoxin. We propose the name VapX _Hi for the HI0451 antitoxin protein. Originally identified on plasmids, TA loci have been found on the chromosomes of a number of bacterial pathogens, and have been implicated in the control of translation during stressful conditions. Translation arrest would enhance survival within human cells and facilitate persistent or chronic mucosal infections.     

Enhanced 1,3-propanediol production in recombinant <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> carrying the gene <Emphasis Type="Italic">yqh</Emphasis>D encoding 1,3-propanediol oxidoreductase isoenzyme

The yqhD gene from Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme (PDORI) and the tetracycline resistant gene (tetR) from plasmid pHY300PLK were amplified by PCR. They were inserted into vector pUC18, yielding the recombinant expression vector pUC18-yqhD-tetR. The recombinant vector was then cloned into Klebsiella pneumoniae ME-308. The overexpression of PDORI in K. pneumoniae surprisingly led to higher 1,3-propanediol production. The final 1,3-propanediol concentration of recombinant K. pneumoniae reached 67.6 g/l, which was 125.33% of that of the original strain. The maximum activity of recombinant PDORI converting 3-HPA to 1,3-PD reached 110 IU/mg after induction by IPTG at 31°C during the fermentation, while it was only 11 IU/mg under the same conditions for the wild type strain. The K _m values of the purified PDORI for 1,3-propanediol and NADP were 12.1 mM and 0.15 mM, respectively. Compared with the original strains, the concentration of the toxic intermediate 3-hydroxypropionaldehyde during the fermentation was also reduced by 22.4%. Both the increased production of 1,3-propanediol and the reduction of toxic intermediate confirmed the significant role of 1,3-propanediol oxidoreductase isoenzyme from E. coli in converting 3-hydroxypropionaldehyde to 1,3-propanediol for 1,3-PD production.     

Gene gain and loss events in <Emphasis Type="Italic">Rickettsia</Emphasis> and <Emphasis Type="Italic">Orientia</Emphasis>species

Background  

Genome degradation is an ongoing process in all members of the Rickettsiales order, which makes these bacterial species an excellent model for studying reductive evolution through interspecies variation in genome size and gene content. In this study, we evaluated the degree to which gene loss shaped the content of some Rickettsiales genomes. We shed light on the role played by horizontal gene transfers in the genome evolution of Rickettsiales.     

Molecular characterization of the <Emphasis Type="Italic">singed wings</Emphasis> locus of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background  

Hormones frequently guide animal development via the induction of cascades of gene activities, whose products further amplify an initial hormonal stimulus. In Drosophila the transformation of the larva into the pupa and the subsequent metamorphosis to the adult stage is triggered by changes in the titer of the steroid hormone 20-hydroxyecdysone. singed wings (swi) is the only gene known in Drosophila melanogaster for which mutations specifically interrupt the transmission of the regulatory signal from early to late ecdysone inducible genes.     

Possible use of <Emphasis Type="Italic">ail</Emphasis> and <Emphasis Type="Italic">foxA</Emphasis> polymorphisms for detecting pathogenic <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis>

Background  

Yersinia enterocolitica is an enteric pathogen that invades the intestinal mucosa and proliferates within the lymphoid follicles (Peyer's patches). The attachment invasion locus (ail) mediates invasion by Y. enterocolitica and confers an invasive phenotype upon non-invasive E. coli; ail is the primary virulence factor of Y. enterocolitica. The ferrioxamine receptor (foxA) located on the Y. enterocolitica chromosome, together with its transport protein, transports a siderophore specific for ferric ion. Currently, ail is the primary target gene for nucleic acid detection of pathogenic Y. enterocolitica.     

Potential for <Emphasis Type="Italic">luxS</Emphasis> related signalling in marine bacteria and production of autoinducer-2 in the genus <Emphasis Type="Italic">Shewanella</Emphasis>

Background  

The autoinducer-2 (AI-2) group of signalling molecules are produced by both Gram positive and Gram negative bacteria as the by-product of a metabolic transformation carried out by the LuxS enzyme. They are the only non species-specific quorum sensing compounds presently known in bacteria. The luxS gene coding for the AI-2 synthase enzyme was found in many important pathogens. Here, we surveyed its occurrence in a collection of 165 marine isolates belonging to abundant marine phyla using conserved degenerated PCR primers and sequencing of selected positive bands to determine if the presence of the luxS gene is phylogenetically conserved or dependent on the habitat.     

Structure and evolution of <Emphasis Type="Italic">Apetala3</Emphasis>, a sex-linked gene in <Emphasis Type="Italic">Silene latifolia</Emphasis>

Background  

The evolution of sex chromosomes is often accompanied by gene or chromosome rearrangements. Recently, the gene AP3 was characterized in the dioecious plant species Silene latifolia. It was suggested that this gene had been transferred from an autosome to the Y chromosome.     

Recombinant production of <Emphasis Type="Italic">Streptococcus equisimilis</Emphasis> streptokinase by <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Background  

Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat) pathway.