Characterization of the <Emphasis Type="Italic">codY</Emphasis> gene and its influence on biofilm formation in <Emphasis Type="Italic">Bacillus </Emphasis><Emphasis Type="Italic">cereus</Emphasis>

The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant (M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation. Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production of the protease (∼130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type. Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels CodY represses production of an unknown protease and is involved in biofilm formation.     

Growth phase-differential quorum sensing regulation of anthranilate metabolism in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas quinolone signal (PQS) plays a role in the regulation of virulence genes and it is intertwined in the las/rhl quorum sensing (QS) circuits of Pseudomonas aeruginosa. PQS is synthesized from anthranilate by pqsA-D and pqsH whose expression is influenced by the las/rhl systems. Since anthranilate can be degraded by functions of antABC and catBCA, PQS synthesis might be regulated by the balance between the expression of the pqsA-D/phnAB, pqsH, antABC, and catBCA gene loci. antA and catA are repressed by LasR during log phase and activated by RhlR in late stationary phase, whereas pqsA-E/phnAB is activated by LasR in log phase and repressed by RhlR. QscR represses both but each repression occurs in a different growth phase. This growth phase-differential regulation appears to be accomplished by the antagonistic interplay of LasR, RhlR, and QscR, mediated by two intermediate regulators, AntR and PqsR, and their cofactors, anthranilate and PQS, where the expressions of antR and pqsR and the production of anthranilate and PQS are growth phase-differentially regulated by QS systems. Especially, the anthranilate level increases in an RhlR-dependent manner at late stationary phase. From these results, we suggest that RhlR and LasR regulate the anthranilate metabolism in a mutually antagonistic and growth phase-differential manner by affecting both the expressions and activities of AntR and PqsR, and that QscR also phase-differentially represses both LasR and RhlR functions in this regulation.     

Urovirulence of<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>: Planktonic cells<Emphasis Type="Italic">vs.</Emphasis> biofilm cells

Planktonic and biofilm cells of a clinical urinary isolate of P. aeruginosa were compared in vitro for their ability to adhere to uroepithelial cells, interaction with macrophages, and for production of virulence factors like extracellular proteinase, elastase, hemolysin, phospholipase C and pyochelin. Biofilm cells showed increased adherence to UECs, which was coupled with reduced uptake and intracellular killing by macrophages. Overall there was a decrease in production of extracellular products by biofilm cells. Comparing the two cell forms for their ability to establish infection in an ascending model of acute pyelonephritis, significant enhancement of renal bacterial load, as well as more pronounced renal pathology developed with biofilm cells.     

Genes related to chromate resistance by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1

Chromate-hypersensitive mutants of the Pseudomonas aeruginosa PAO1 strain were isolated using transposon-insertion mutagenesis. Comparison of the nucleotide sequences of the regions interrupted in the mutants with the PAO1 genome revealed that the genes affected in three mutant strains were oprE (ORF PA0291), rmlA (ORF PA5163), and ftsK (ORF PA2615), respectively. A relationship of these genes with chromate tolerance has not been previously reported. No other phenotypic changes were observed in the oprE mutant but its resistance to chromate was not fully restored by expressing the ChrA protein, which extrudes chromate ions from the cytoplasm to the periplasmic space. These data suggest that OprE participates in the efflux of chromate from the periplasm to the outside. Increased susceptibility of the rmlA mutant to the metals cadmium and mercury and to the anion-superoxide generator paraquat suggests a protective role of LPS against chromate toxicity. A higher susceptibility of the ftsK mutant to compounds affecting DNA structure (ciprofloxacin, tellurite, mitomycin C) suggests a role of FtsK in the recombinational repair of DNA damage caused by chromate. In conclusion, the P. aeruginosa genome contains diverse genes related to its intrinsic resistance to chromate. Systems pertaining to the outer membrane (OprE), the cell wall (LPS), and the cytoplasm (FtsK) were identified in this work as involved in chromate protection mechanisms.     

Heterologous expression of human paraoxonases in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> inhibits biofilm formation and decreases antibiotic resistance

Quorum sensing (QS) regulates virulence and biofilm formation in Pseudomonas aeruginosa and other medically relevant bacteria. Human paraoxonases (hPONs) are a family of closely related enzymes with multiple functions, including inactivation of the QS signal molecule in P. aeruginosa. However, there is no direct evidence to show the functions of hPONs on biofilm formation and antibiotic resistance in P. aeruginosa. In the present study, hPONs (hPON1, hPON2, and hPON3) genes were respectively cloned into the pMEKm12 shuttle vector and transformed into P. aeruginosa strain PAO1. Expression of the three recombinant proteins was confirmed by Western blotting, and growth of the recombinant strains was not affected by the hPONs gene expression. Biofilm formation and antibiotics resistance of the hPONs recombinant strains were analyzed. Our results showed that biofilm formation was significantly inhibited in all of the three hPONs recombinant strains. Interestingly, this inhibition can be reverted by addition of the corresponding hPONs polyclonal antibodies in the culture media, further indicating that the inhibition of biofilm formation was due to hPONs protein expression. In addition, we also demonstrated that hPONs expression decreased resistance of P. aeruginosa to gentamicin and ceftazidima, two antibiotics clinically used for the treatment of P. aeruginosa infection.     

Betulin inhibits cariogenic properties of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> by targeting <Emphasis Type="Italic">vicRK</Emphasis> and <Emphasis Type="Italic">gtf</Emphasis> genes

Streptococcus mutans, a multivirulent pathogen is considered the primary etiological agent in dental caries. Development of antibiotic resistance in the pathogen has created a need for novel antagonistic agents which can control the virulence of the organism and reduce resistance development. The present study demonstrates the in vitro anti-virulence potential of betulin (lup-20(29)-ene-3β,28-diol), an abundantly available plant triterpenoid against S. mutans UA159. Betulin exhibited significant dose dependent antibiofilm activity without affecting bacterial viability. At 240 µg/ml (biofilm inhibitory concentration), betulin inhibited biofilm formation and adherence to smooth glass surfaces by 93 and 71 % respectively. It reduced water insoluble glucan synthesis by 89 %, in conjunction with down regulation of gtfBC genes. Microscopic analysis confirmed the disruption in biofilm architecture and decreased exopolysaccharide production. Acidogenicity and aciduricity, key virulence factors responsible for carious lesions, were also notably affected. The induced auto-aggregation of cells upon treatment could be due to the down regulation of vicK. Results of gene expression analysis demonstrated significant down-regulation of virulence genes upon betulin treatment. Furthermore, the nontoxic effect of betulin on peripheral blood mononuclear cells even after 72 h treatment makes it a strong candidate for assessing its suitability to be used as a therapeutic agent.     

Formation of <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> biofilms on multiple surfaces on <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Summary A liquid-based assay was used to evaluate the ability of Yersinia pseudotuberculosis to form a bacterial biofilm on the nematode Caenorhabditis elegans. After 3 days of incubation in the liquid assay a biofilm was clearly visible by light microscopy on both the head and vulva region of the worms. At times, the biofilm formation on the vulva appeared to prevent the laying of eggs by the adult hermaphrodite; the eggs would later hatch inside of the worm. One possible explanation for the biofilm formation observed on the vulva may be the increased motion of the cuticle surrounding the vulva when the worm is immersed in a liquid culture. This is the first report of biofilm formation on the vulva of C. elegans.     

Antipathogenic potential of marine <Emphasis Type="Italic">Bacillus</Emphasis> sp. SS4 on <Emphasis Type="Italic">N</Emphasis>-acyl-homoserine-lactone-mediated virulence factors production in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> (PAO1)

Antipathogenic therapy is an outcome of the quorum-sensing inhibition (QSI) mechanism, which targets autoinducer-dependent virulent gene expression in bacterial pathogens. N-acyl homoserine lactone (AHL) acts as a key regulator in the production of virulence factors and biofilm formation in Pseudomonas aeruginosa PAO1 and violacein pigment production in Chromobacterium violaceum. In the present study, the marine bacterial strain SS4 showed potential QSI activity in a concentration-dependent manner (0.5–2 mg/ml) against the AHL-mediated violacein production in C. violaceum (33–86%) and biofilm formation (33–88%), total protease (20–65%), LasA protease (59–68%), LasB elastase (36–68%), pyocyanin (17–86%) and pyoverdin productions in PAO1. The light and confocal laser scanning microscopic analyses confirmed the reduction of the biofilm-forming ability of PAO1 when treated with SS4 extract. Furthermore, the antibiofilm potential was confirmed through static biofilm ring assay, in which ethyl acetate extract of SS4 showed concentration-dependent reduction in the biofilm-forming ability of PAO1. Thus, the result of this study clearly reveals the antipathogenic and antibiofilm properties of the bacterial isolate SS4. Through 16S rDNA analysis, the strain SS4 was identified as Bacillus sp. (GenBank Accession Number: GU471751).     

The Evolutionary History of <Emphasis Type="Italic">Shigella</Emphasis> and Enteroinvasive <Emphasis Type="Italic">Escherichia coli</Emphasis> Revised

In Shigella and enteroinvasive Escherichia coli (EIEC), the etiologic agents of shigellosis in humans, the determinants responsible for entry of bacteria into and dissemination within epithelial cells are encoded by a virulence plasmid. To understand the evolution of the association between the virulence plasmid and the chromosome, we performed a phylogenetic analysis using the sequences of four chromosomal genes (trpA, trpB, pabB, and putP) and three virulence plasmid genes (ipaB, ipaD, and icsA) of a collection of 51 Shigella and EIEC strains. The phylogenetic tree derived from chromosomal genes showed a typical star phylogeny, indicating a fast diversification of Shigella and EIEC groups. Phylogenetic groups obtained from the chromosomal and plasmidic genes were similar, suggesting that the virulence plasmid and the chromosome share similar evolutionary histories. The few incongruences between the trees could be attributed to exchanges of fragments of different plasmids and not to the transfer of an entire plasmid. This indicates that the virulence plasmid was not transferred between the different Shigella and EIEC groups. These data support a model of evolution in which the acquisition of the virulence plasmid in an ancestral E. coli strain preceded the diversification by radiation of all Shigella and EIEC groups, which led to highly diversified but highly specialized pathogenic groups.     

<Emphasis Type="Italic">WUS</Emphasis> and <Emphasis Type="Italic">STM</Emphasis>-based reporter genes for studying meristem development in poplar

We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5′ flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50–60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common in other organs, including in leaf veins (40 and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems as explants showed that expression was detectable prior to visible shoot development, starting 3–15 days after explants were placed onto callus inducing medium. A minority of WUS and STM events also showed expression in the cambium, phloem, or xylem of regenerated, greenhouse grown plants undergoing secondary growth. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Both paralogs of poplar STM were down-regulated threefold to sixfold during early callus initiation. We identified 15–35 copies of cytokinin response regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the events recovered may be useful for studying the process of primary and secondary meristem development, including treatments intended to stimulate meristem development to promote clonal propagation and genetic transformation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.