A novel pathway producing dimethylsulphide in bacteria is widespread in soil environments

The volatile compound dimethylsulphide (DMS) is important in climate regulation, the sulphur cycle and signalling to higher organisms. Microbial catabolism of the marine osmolyte dimethylsulphoniopropionate (DMSP) is thought to be the major biological process generating DMS. Here we report the discovery and characterisation of the first gene for DMSP-independent DMS production in any bacterium. This gene, mddA, encodes a methyltransferase that methylates methanethiol (MeSH) and generates DMS. MddA functions in many taxonomically diverse bacteria including sediment-dwelling pseudomonads, nitrogen-fixing bradyrhizobia and cyanobacteria, and mycobacteria, including the pathogen Mycobacterium tuberculosis. The mddA gene is present in metagenomes from varied environments, being particularly abundant in soil environments, where it is predicted to occur in up to 76% of bacteria. This novel pathway may significantly contribute to global DMS emissions, especially in terrestrial environments, and could represent a shift from the notion that DMSP is the only significant precursor of DMS

Genetic and biochemical analyses of chromosome and plasmid gene homologues encoding ICL and ArCP domains in Vibrioanguillarum strain 775

Anguibactin, the siderophore produced by Vibrio anguillarum 775 is synthesized from 2,3-dihydroxybenzoic acid (DHBA), cysteine and hydroxyhistamine via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes encoding anguibactin biosynthetic proteins are harbored by the pJM1 plasmid. In this work we report the identification of a homologue of the plasmid-encoded angB on the chromosome of strain 775. The product of both genes harbor an isochorismate lyase (ICL) domain that converts isochorismic acid to 2,3-dihydro-2,3-dihydroxybenzoic acid, one of the steps of DHBA synthesis. We show in this work that both ICL domains are functional in the production of DHBA in V. anguillarum as well as in E. coli. Substitution by alanine of the aspartic acid residue in the active site of both ICL domains completely abolishes their isochorismate lyase activity in vivo. The two proteins also carry an aryl carrier protein (ArCP) domain. In contrast with the ICL domains only the plasmid encoded ArCP can participate in anguibactin production as determined by complementation analyses and site-directed mutagenesis in the active site of the plasmid encoded protein, S248A. The site-directed mutants, D37A in the ICL domain and S248A in the ArCP domain of the plasmid encoded AngB were also tested in vitro and clearly show the importance of each residue for the domain function and that each domain operates independently.

The stealth episome: suppression of gene expression on the excised genomic island PPHGI-1 from Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in the common bean, Phaseolus vulgaris. P. syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene avrPphB (syn. hopAR1), which resides on PPHGI-1, a 106 kb genomic island. Loss of PPHGI-1 from P. syringae pv. phaseolicola 1302A following exposure to the hypersensitive resistance response (HR) leads to the evolution of strains with altered virulence. Here we have used fluorescent protein reporter systems to gain insight into the mobility of PPHGI-1. Confocal imaging of dual-labelled P. syringae pv. phaseolicola 1302A strain, F532 (dsRFP in chromosome and eGFP in PPHGI-1), revealed loss of PPHGI-1::eGFP encoded fluorescence during plant infection and when grown in vitro on extracted leaf apoplastic fluids. Fluorescence-activated cell sorting (FACS) of fluorescent and non-fluorescent PPHGI-1::eGFP F532 populations showed that cells lost fluorescence not only when the GI was deleted, but also when it had excised and was present as a circular episome. In addition to reduced expression of eGFP, quantitative PCR on sub-populations separated by FACS showed that transcription of other genes on PPHGI-1 (avrPphB and xerC) was also greatly reduced in F532 cells harbouring the excised PPHGI-1::eGFP episome. Our results show how virulence determinants located on mobile pathogenicity islands may be hidden from detection by host surveillance systems through the suppression of gene expression in the episomal state

Use of genetically modified bacteria for drug delivery in humans: Revisiting the safety aspect

The use of live, genetically modified bacteria as delivery vehicles for biologics is of considerable interest scientifically and has attracted significant commercial investment. We have pioneered the use of the commensal gut bacterium Bacteroides ovatus for the oral delivery of therapeutics to the gastrointestinal tract. Here we report on our investigations of the biological safety of engineered B. ovatus bacteria that includes the use of thymineless death as a containment strategy and the potential for the spread of transgenes in vivo in the mammalian gastrointestinal tract. We demonstrate the ability of GM-strains of Bacteroides to survive thymine starvation and overcome it through the exchange of genetic material. We also provide evidence for horizontal gene transfer in the mammalian gastrointestinal tract resulting in transgene-carrying wild type bacteria. These findings sound a strong note of caution on the employment of live genetically modified bacteria for the delivery of biologics

The luxS mutation causes loosely-bound biofilms in Shewanella oneidensis

<p>Abstract</p> <p>Background</p> <p>The <it>luxS </it>gene in <it>Shewanella oneidensis </it>was shown to encode an autoinducer-2 (AI-2)-like molecule, the postulated universal bacterial signal, but the impaired biofilm growth of a <it>luxS </it>deficient mutant could not be restored by AI-2, indicating it might not have a signalling role in this organism.</p> <p>Findings</p> <p>Here, we provide further evidence regarding the metabolic role of a <it>luxS </it>mutation in <it>S. oneidensis</it>. We constructed a <it>luxS </it>mutant and compared its phenotype to a wild type control with respect to its ability to remove AI-2 from the medium, expression of secreted proteins and biofilm formation. We show that <it>S. oneidensis </it>has a cell-dependent mechanism by which AI-2 is depleted from the medium by uptake or degradation at the end of the exponential growth phase. As AI-2 depletion is equally active in the <it>luxS </it>mutant and thus does not require AI-2 as an inducer, it appears to be an unspecific mechanism suggesting that AI-2 for <it>S. oneidensis </it>is a metabolite which is imported under nutrient limitation. Secreted proteins were studied by iTraq labelling and liquid chromatography mass spectrometry (LC-MS) detection. Differences between wild type and mutant were small. Proteins related to flagellar and twitching motility were slightly up-regulated in the <it>luxS </it>mutant, in accordance with its loose biofilm structure. An enzyme related to cysteine metabolism was also up-regulated, probably compensating for the lack of the LuxS enzyme. The <it>luxS </it>mutant developed an undifferentiated, loosely-connected biofilm which covered the glass surface more homogenously than the wild type control, which formed compact aggregates with large voids in between.</p> <p>Conclusions</p> <p>The data confirm the role of the LuxS enzyme for biofilm growth in <it>S. oneidensis </it>and make it unlikely that AI-2 has a signalling role in this organism.</p

Cloning and Characterization of Genes Involved in Nostoxanthin Biosynthesis of Sphingomonas elodea ATCC 31461

Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB) and a β-carotene hydroxylase gene (crtZ) located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,2′-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of β-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and β-carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,2′-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed

Stability, Entrapment and Variant Formation of Salmonella Genomic Island 1

<div><h3>Background</h3><p>The <em>Salmonella</em> genomic island 1 (SGI1) is a 42.4 kb integrative mobilizable element containing several antibiotic resistance determinants embedded in a complex integron segment In104. The numerous SGI1 variants identified so far, differ mainly in this segment and the explanations of their emergence were mostly based on comparative structure analyses. Here we provide experimental studies on the stability, entrapment and variant formation of this peculiar gene cluster originally found in <em>S</em>. Typhimurium.</p> <h3>Methodology/Principal Findings</h3><p>Segregation and conjugation tests and various molecular techniques were used to detect the emerging SGI1 variants in <em>Salmonella</em> populations of 17 <em>Salmonella enterica</em> serovar Typhimurium DT104 isolates from Hungary. The SGI1s in these isolates proved to be fully competent in excision, conjugal transfer by the IncA/C helper plasmid R55, and integration into the <em>E. coli</em> chromosome. A trap vector has been constructed and successfully applied to capture the island on a plasmid. Monitoring of segregation of SGI1 indicated high stability of the island. SGI1-free segregants did not accumulate during long-term propagation, but several SGI1 variants could be obtained. Most of them appeared to be identical to SGI1-B and SGI1-C, but two new variants caused by deletions via a short-homology-dependent recombination process have also been detected. We have also noticed that the presence of the conjugation helper plasmid increased the formation of these deletion variants considerably.</p> <h3>Conclusions/Significance</h3><p>Despite that excision of SGI1 from the chromosome was proven in SGI1⁺<em>Salmonella</em> populations, its complete loss could not be observed. On the other hand, we demonstrated that several variants, among them two newly identified ones, arose with detectable frequencies in these populations in a short timescale and their formation was promoted by the helper plasmid. This reflects that IncA/C helper plasmids are not only involved in the horizontal spreading of SGI1, but may also contribute to its evolution.</p> </div

GFP-tagged multimetal-tolerant bacteria and their detection in the rhizosphere of white mustard

The introduction of rhizobacteria that tolerate heavy metals is a promising approach to support plants involved in phytoextraction and phytostabilisation. In this study, soil of a metal-mine wasteland was analyzed for the presence of metal-tolerant bacterial isolates, and the tolerance patterns of the isolated strains for a number of heavy metals and antibiotics were compared. Several of the multimetal-tolerant strains were tagged with a broad host range reporter plasmid (i.e. pPROBE-NT) bearing a green fluorescent protein marker gene (gfp). Overall, the metal-tolerant isolates were predominately Gram-negative bacteria. Most of the strains showed a tolerance to five metals (Zn, Cu, Ni, Pb and Cd), but with differing tolerance patterns. From among the successfully tagged isolates, we used the transconjugant Pseudomonas putida G25 (pPROBE-NT) to inoculate white mustard seedlings. Despite a significant decrease in transconjugant abundance in the rhizosphere, the gfp-tagged cells survived on the root surfaces at a level previously reported for root colonisers