In people with type 2 diabetes (T2D), the presence of microvascular disease is associated with increased risk for macrovascular events [(1)][1]. Of particular interest is whether microvascular disease is a risk factor for heart failure (HF) or has a role in its pathophysiology [(2)][2]. The

Aims:To characterize atrazine-degrading potential of bacterial communities enriched from agrochemical factory soil by analysing diversity and organization of catabolic genes.Methods and Results:The bacterial communities enriched from three different sites of varying atrazine contamination mineralized 65-80% of 14C ring-labelled atrazine. The presence of trzN-atzBC-trzD, trzN-atzABC-trzD and trzN-atzABCDEF-trzD gene combinations was determined by PCR. In all enriched communities, trzN-atzBC genes were located on a 165-kb plasmid, while atzBC or atzC genes were located on separated plasmids. Quantitative PCR revealed that catabolic genes were present in up to 4% of the community. Restriction analysis of 16S rDNA clone libraries of the three enrichments revealed marked differences in microbial community structure and diversity. Sequencing of selected clones identified members belonging to Proteobacteria (alpha-, beta- and gamma-subclasses), the Actinobacteria, Bacteroidetes and TM7 division. Several 16S rRNA gene sequences were closely related to atrazine-degrading community members previously isolated from the same contaminated site.Conclusions:The enriched communities represent a complex and diverse bacterial associations displaying heterogeneity of catabolic genes and their functional redundancies at the first steps of the upper and lower atrazine-catabolic pathway. The presence of catabolic genes in small proportion suggests that only a subset of the community has the capacity to catabolize atrazine.Significance and Impact of the Study:This study provides insights into the genetic specificity and the repertoire of catabolic genes within bacterial communities originating from soils exposed to long-term contamination by s-triazine compounds.

Aims: To characterize an atrazine-degrading bacterial community enriched from the wastewater of a herbicide factory.Methods and Results: The community mineralized 81.4 +/- 1.9% of [C-14-ring]atrazine and 31.0 +/- 1.8% of [C-14-ethyl]atrazine within 6 days of batch cultivation in mineral salts medium containing atrazine as the sole nitrogen source. Degradation activity of the community towards different chloro- and methylthio-substituted s-triazine compounds was also demonstrated. Restriction analysis of amplified 16S rDNA revealed high diversity of bacterial populations forming the community, with Pseudomonas species dominating in the clone library. Atrazine-degrading genetic potential of the community determined by PCR revealed the presence of trzN, atzB, atzC and trzD genes. The trzN, atzB and atzC genes were shown to be located on a plasmid of 322 kb. Quantitative PCR showed that relative abundances of atzB, atzC and trzD genes were approx. 100-fold lower than 16S rDNA.Conclusions: The enriched community represents a complex bacterial association expressing substantial atrazine-mineralizing activity and a broad specificity towards a range of s-triazine compounds.Significance and Impact of the Study: Our study is beginning to yield insights into the richness, genetic potential and density of functional atrazine-mineralizing community that could be a potential bioaugmentation agent for improving biotransformation processes in wastewaters bearing different s-triazine compounds.

The plasmid pADP1::Tn5 derived from pADP1[Atr+] carrying a Tn5 transposon conferring kanamycin and streptomycin resistances was constructed and introduced in Agrobacterium tumefaciens St96-4. This genetically modified strain was inoculated (approximately 10(8) cfu g(-1)) in potted soils planted with maize and treated or not with atrazine (1.5 mg kg(-1)). Bulk and maize rhizosphere soils were sampled 39 days after planting to look for soil indigenous bacteria that had acquired pADP1::Tn5. Four transconjugants were isolated from four different soil samples. The estimated transfer frequency of pADP1::Tn5 was 10(-4) per donor. Maize rhizosphere and atrazine treatment had no obvious effect on pADP1::Tn5 transfer frequency. The sequencing of the 16S rDNA sequences of the transconjugants revealed that they were almost identical and highly similar to that of Variovorax spp (97%). In addition, their characterization suggested that the atzA and atzB genes had been transferred from pADP1::Tn5 to the bacterial chromosome in two of the four transconjugants. These data suggest that the atz degrading genes are horizontally transferred in soil and possibly subjected to gene rearrangement.

The level of expression of highly conserved, plasmid-borne, and widely dispersed atrazine catabolic genes (atz) was studied by RT-qPCR in two telluric atrazine-degrading microbes. RT-qPCR assays, based on the use of real-time PCR, were developed in order to quantify atzABCDEF mRNAs in Pseudomonas sp. ADP and atzABC mRNAs in Chelatobacter heintzii. atz gene expression was expressed as mRNA copy number per 10(6) 16S rRNA. In Pseudomonas sp. ADP, atz genes were basally expressed. It confirmed atrazine-degrading kinetics indicating that catabolic activity starts immediately after adding the herbicide. atz gene expression increased transitorily in response to atrazine treatment. This increase was only observed while low amount of atrazine remained in the medium. In C. heintzii, only atzA was basally expressed. atzA and atzB expression levels were similarly and significantly increased in response to atrazine treatment. atzC was not expressed even in the presence of high amounts of atrazine. This study showed that atz genes are basally expressed and up-regulated in response to atrazine treatment. atz gene expression patterns are different in Pseudomonas ADP and C. heintzii suggesting that the host may influence the expression of plasmid-borne atrazine-catabolic potential.