Thursday, September 20, 2012

Phenylacetic acid catabolism in Corynebacterium glutamicum

Corynebacterium glutamicum AS 1.542 is an important industrial bacterium that metabolizes aromatic compounds, breaking them down into digestible molecules that are suitable carbon sources for growth. Chen, et al. describes the elucidation of the breakdown of phenylacetic acid into ready metabolites that feed into the Kreb’s cycle. They further describe the finding that not all strains of the C. glutamicum species, such as strain ATCC 13032, are able to perform this function but observe that it is a transferable characteristic that can be achieved when transformed with a recombinant plasmid containing the operon that encodes the genes of the operon.

Mapping of transcription start sites was accomplished using a novel adaptation of RNA 5’-Polyphosphatase, Terminator™ 5’-Phosphate-Dependent Exonuclease and Ribo-Zero™ rRNA Removal Kit (Gram- Positive Bacteria). Briefly, total RNA was isolated from C. glutamicum strain AS 1.542, and followed by use of the Ribo-Zero rRNA Removal kit.  Following the rRNA depletion, the remaining cellular RNA was digested with Terminator Exonuclease to remove any unwanted 5’-monophosphosphorylated RNA.  Following cleanup, the remaining RNA was then further treated with RNA-5’-Polyphosphatase to generate 5’-monophosphates on the bacterial mRNA that are suitable for RNA-Seq library production. The remaining RNA was fragmented and size selected into fragments of 200-500 bases. The RNA, now with “enriched” 5’-ends, was  used to create a small RNA-Seq library using Illumina’s Tru-Seq™ Small RNA Library Preparation Kit, and sequenced on an Illumina GAIIx sequencer.  The resulting reads were aligned to the paa gene cluster in the C. glutamium reference genome sequence. By combining published information about promoter regions in C. glutamicum with 5-end enriched RNAseq data, probable transcription start sites and promoter regions were deduced for the paa gene cluster.

ResearchBlogging.orgChen X et al (2012). Phenylacetic Acid Catabolism and Its Transcriptional Regulation in Corynebacterium glutamicum. Applied and environmental microbiology, 78 (16), 5796-804 PMID: 22685150

Friday, September 7, 2012

Transposome-mediated validation of essential genes in Acinetobacter baumannii

Umland et al. investigated essential genes for growth in Acinetobacter baumannii by generating mutants using the EZ-Tn5™ KAN-2 Transposome. In the past, other researchers have found EZ-Tn5 transposomes extremely useful in characterizing the genetics of this bacterium.

A subset of 34 mutants with unique gene disruptions that demonstrated little to no growth on ascites underwent evaluation in a rat subcutaneous abscess model, and these results established that 18 (53%) of these genes could be classed as “in vivo essential”. The putative gene products all had known biological functions, and represented potentially untested, unrecognized, or underexploited targets for antibiotics that could be useful in treating infections in a living host. These genes could be classified into five functional categories: metabolic, two-component signaling systems, DNA/RNA synthesis and regulation, protein transport, and structural. These A. baumannii in vivo essential genes overlapped poorly with the sets of essential genes from other Gram-negative bacteria in the Database of Essential Genes (DEG), including those of Acinetobacter baylyi, a closely related species.

None of the 18 in vivo essential genes identified in this study, or their putative gene products, were classed as targets for currently existing antibiotics. The findings indicate that potentially useful antimicrobials may be developed that will be effective in treating Acinetobacter (or other) infections, though the researchers state that at this time there do not appear to be any FDA-approved antimicrobials or any other drugs in the R&D pipeline that target the newly identified genes.

ResearchBlogging.orgUmland TC et al. (2012). In Vivo-Validated Essential Genes Identified in Acinetobacter baumannii by Using Human Ascites Overlap Poorly with Essential Genes Detected on Laboratory Media. mBio, 3 (4) PMID: 22911967