Tunable diode laser absorption spectroscopy as method of choice for non-invasive and automated detection of microbial growth in media fills.

Tunable diode laser absorption spectroscopy (TDLAS) was evaluated on its potential to detect bacterial growth of contaminated media fill vials. The target was a replacement/ automation of the traditional visual media fill inspection. TDLAS was used to determine non-invasively O2 and/or CO2 changes in headspaces of such vials being induced by metabolically active microorganisms. Four different vial formats, 34 microorganisms (inoculation volume<10 cells) and two different media (TSB/FTM) were tested. Applying parallel CO2 and O2 headspace measurements all format-organism combinations were detected within <11 days reliably with reproducible results. False negatives were exclusively observed for samples that were intentionally breached with syringes of 0.3mm in diameter. Overall it was shown that TDLAS functionality for a replacement of the visual media fill inspection is given and that investing in further validation and implementation studies is valuable. Nevertheless, some small but vincible challenges remain to have this technology in practical use.

The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus.

Homologous recombination (HR) is crucial for maintaining genome integrity by repairing DNA double-strand breaks (DSBs) and rescuing collapsed replication forks. In contrast, uncontrolled HR can lead to chromosome translocations, loss of heterozygosity, and deletion of repetitive sequences. Controlled HR is particularly important for the preservation of repetitive sequences of the ribosomal gene (rDNA) cluster. Here we show that recombinational repair of a DSB in rDNA in Saccharomyces cerevisiae involves the transient relocalization of the lesion to associate with the recombination machinery at an extranucleolar site. The nucleolar exclusion of Rad52 recombination foci entails Mre11 and Smc5-Smc6 complexes and depends on Rad52 SUMO (small ubiquitin-related modifier) modification. Remarkably, mutations that abrogate these activities result in the formation of Rad52 foci within the nucleolus and cause rDNA hyperrecombination and the excision of extrachromosomal rDNA circles. Our study also suggests a key role of sumoylation for nucleolar dynamics, perhaps in the compartmentalization of nuclear activities.

Control of Rad52 recombination activity by double-strand break-induced SUMO modification.

Homologous recombination is essential for genetic exchange, meiosis and error-free repair of double-strand breaks. Central to this process is Rad52, a conserved homo-oligomeric ring-shaped protein, which mediates the exchange of the early recombination factor RPA by Rad51 and promotes strand annealing. Here, we report that Rad52 of Saccharomyces cerevisiae is modified by the ubiquitin-like protein SUMO, primarily at two sites that flank the conserved Rad52 domain. Sumoylation is induced on DNA damage and triggered by Mre11-Rad50-Xrs2 (MRX) complex-governed double-strand breaks (DSBs). Although sumoylation-defective Rad52 is largely recombination proficient, mutant analysis revealed that the SUMO modification sustains Rad52 activity and concomitantly shelters the protein from accelerated proteasomal degradation. Furthermore, our data indicate that sumoylation becomes particularly relevant for those Rad52 molecules that are engaged in recombination.

Identification of SUMO-protein conjugates.

Modification of proteins by covalent attachment of ubiquitin and the ubiquitin-like modifier SUMO are widespread regulatory events of all eukaryotic cells. SUMOylation has received much attention, because several identified targets play prominent roles, in particular, in cell signaling, gene expression, and DNA repair. Notably, only a very small fraction of a substrate is usually SUMOylated at steady-state levels, which could be because modification is reversible and transient. Because of the low level of modification, SUMOylated proteins are often overlooked or sometimes misinterpreted as a less important fraction of a protein pool. Here we discuss procedures that can circumvent identification problems and describe methods for their verification.

SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase.

Damaged DNA, if not repaired before replication, can lead to replication fork stalling and genomic instability; however, cells can switch to different damage bypass modes that permit replication across lesions. Two main bypasses are controlled by ubiquitin modification of proliferating cell nuclear antigen (PCNA), a homotrimeric DNA-encircling protein that functions as a polymerase processivity factor and regulator of replication-linked functions. Upon DNA damage, PCNA is modified at the conserved lysine residue 164 by either mono-ubiquitin or a lysine-63-linked multi-ubiquitin chain, which induce error-prone or error-free replication bypasses of the lesions. In S phase, even in the absence of exogenous DNA damage, yeast PCNA can be alternatively modified by the small ubiquitin-related modifier protein SUMO; however the consequences of this remain controversial. Here we show by genetic analysis that SUMO-modified PCNA functionally cooperates with Srs2, a helicase that blocks recombinational repair by disrupting Rad51 nucleoprotein filaments. Moreover, Srs2 displays a preference for interacting directly with the SUMO-modified form of PCNA, owing to a specific binding site in its carboxy-terminal tail. Our finding suggests a model in which SUMO-modified PCNA recruits Srs2 in S phase in order to prevent unwanted recombination events of replicating chromosomes.

A unique hydrophobic cluster near the active site contributes to differences in borrelidin inhibition among threonyl-tRNA synthetases.

Borrelidin, a compound with anti-microbial and anti-angiogenic properties, is a known inhibitor of bacterial and eukaryal threonyl-tRNA synthetase (ThrRS). The inhibition mechanism of borrelidin is not well understood. Archaea contain archaeal and bacterial genre ThrRS enzymes that can be distinguished by their sequence. We explored species-specific borrelidin inhibition of ThrRSs. The activity of ThrRS from Sulfolobus solfataricus and Halobacterium sp. NRC-1 was inhibited by borrelidin, whereas ThrRS enzymes from Methanocaldococcus jannaschii and Archaeoglobus fulgidus were not. In Escherichia coli ThrRS, borrelidin binding induced a conformational change, and threonine was not activated as shown by ATP-PP(i) exchange and a transient kinetic assay measuring intrinsic tryptophan fluorescence changes. These assays further showed that borrelidin is a noncompetitive tight binding inhibitor of E. coli ThrRS with respect to threonine and ATP. Genetic selection of borrelidin-resistant mutants showed that borrelidin binds to a hydrophobic region (Thr-307, His-309, Cys-334, Pro-335, Leu-489, Leu-493) proximal to the zinc ion at the active site of the E. coli ThrRS. Mutating residue Leu-489 --> Trp reduced the space of the hydrophobic cluster and resulted in a 1500-fold increase of the K(i) value from 4 nM to 6 microm. An alignment of ThrRS sequences showed that this cluster is conserved in most organisms except for some Archaea (e.g. M. jannaschii, A. fulgidus) and some pathogens (e.g. Helicobacter pylori). This study illustrates how one class of natural product inhibitors affects aminoacyl-tRNA synthetase function, providing potentially useful information for structure-based inhibitor design.

A freestanding proofreading domain is required for protein synthesis quality control in Archaea.

Threonyl-tRNA synthetase (ThrRS) participates in protein synthesis quality control by selectively editing the misacylated species Ser-tRNA(Thr). In bacteria and eukaryotes the editing function of ThrRS resides in a highly conserved N-terminal domain distant from the active site. Most archaeal ThrRS proteins are devoid of this editing domain, suggesting evolutionary divergence of quality-control mechanisms. Here we show that archaeal editing of Ser-tRNAThr is catalyzed by a domain unrelated to, and absent from, bacterial and eukaryotic ThrRSs. Despite the lack of sequence homology, the archaeal and bacterial editing domains are both reliant on a pair of essential histidine residues suggestive of a common catalytic mechanism. Whereas the archaeal editing module is most commonly part of full-length ThrRS, several crenarchaeal species contain individual genes encoding the catalytic (ThrRS-cat) and editing domains (ThrRS-ed). Sulfolobus solfataricus ThrRS-cat was shown to synthesize both Thr-tRNAThr and Ser-tRNAThr and to lack editing activity against Ser-tRNAThr. In contrast, ThrRS-ed lacks aminoacylation activity but can act as an autonomous protein in trans to hydrolyze specifically Ser-tRNAThr, or it can be fused to ThrRS-cat to provide the same function in cis. Deletion analyses indicate that ThrRS-ed is dispensable for growth of S. solfataricus under standard conditions but is required for normal growth in media with elevated serine levels. The growth phenotype of the ThrRS-ed deletion strain suggests that retention of the discontinuous ThrRS quaternary structure relates to specific physiological requirements still evident in certain Archaea.