Diffraction Before Destruction: A bottleneck in x-ray crystallography is the growth of well-ordered crystals large enough to obtain high-resolution diffraction data within an exposure that limits radiation damage. Serial femtosecond crystallography promises to overcome these constraints by using short intense pulses that out-run radiation damage. A stream of crystals is flowed across the free-electron beam and for each pulse, diffraction data is recorded from a single crystal before it is destroyed. Redecke et al. (p. 227 , published online 29 November; see the Perspective by Helliwell ) used this technique to determine the structure of an enzyme from Trypanosoma brucei , the parasite that causes sleeping sickness, from micron-sized crystals grown within insect cells. The structure shows how this enzyme, which is involved in degradation of host proteins, is natively inhibited prior to activation, which could help in the development of parasite-specific inhibitors.

Biological analyses traditionally probe cell ensembles in the range of 10 ³ –10 ⁶ cells, thereby completely averaging over relevant individual cell responses, such as differences in cell proliferation, responses to external stimuli or disease onset. In past years, this fact has been realized and increasing interest has evolved for single-cell analytical methods, which could give exciting new insights into genomics, proteomics, transcriptomics and systems biology. Microfluidic or lab-on-a-chip devices are the method of choice for single-cell analytical tools as they allow the integration of a variety of necessary process steps involved in single-cell analysis, such as selection, navigation, positioning or lysis of single cells as well as separation and detection of cellular analytes. Along with this advantageous integration, microfluidic devices confine single cells in compartments near their intrinsic volume, thus minimizing dilution effects and increasing detection sensitivity. This review overviews the developments and achievements of microfluidic single-cell analysis of intracellular compounds in the past few years, from proof-of-principle devices to applications demonstrating a high biological relevance.

A regulatory network ofSinorhizobium melilotigenes involved in adaptation to iron-limiting conditions and the involvement of the rhizobial iron regulator gene (rirA) were analyzed by mutation and microarray analyses. A constructedS. meliloti rirAmutant exhibited growth defects and enhanced H₂O₂sensitivity in the presence of iron, but symbiotic nitrogen fixation was not affected. To identify iron-responsive and RirA-regulatedS. melilotigenes, a transcriptome approach using whole-genome microarrays was used. Altogether, 45 genes were found to be jointly derepressed by mutation ofrirAand under different iron-limited conditions. As expected, a number of genes involved in iron transport (e.g.,hmuPSTU,shmR,rhbABCDEF,rhtX, andrhtA) and also genes with predicted functions in energy metabolism (e.g.,fixN3,fixP3, andqxtAB) and exopolysaccharide production (e.g.,exoYandexoN) were found in this group of genes. In addition, the iron deficiency response ofS. melilotialso involvedrirA-independent expression changes, including repression of theS. melilotiflagellar regulon. Finally, the RirA modulon also includes genes that are not iron responsive, including a gene cluster putatively involved in Fe-S cluster formation (sufA,sufS,sufD,sufC, andsufB).

We present the first high-resolution determination of transcriptome architecture in the priority pathogen Acinetobacter baumannii. Pooled RNA from 16 laboratory conditions was used for differential RNA-seq (dRNA-seq) to identify 3731 transcriptional start sites (TSS) and 110 small RNAs, including the first identification in A. baumannii of sRNAs encoded at the 3' end of coding genes. Most sRNAs were conserved among sequenced A. baumannii genomes, but were only weakly conserved or absent in other Acinetobacter species. Single nucleotide mapping of TSS enabled prediction of -10 and -35 RNA polymerase binding sites and revealed an unprecedented base preference at position +2 that hints at an unrecognized transcriptional regulatory mechanism. To apply functional genomics to the problem of antimicrobial resistance, we dissected the transcriptional regulation of the drug efflux pump responsible for chloramphenicol resistance, craA. The two craA promoters were both down-regulated >1000-fold when cells were shifted to nutrient limited medium. This conditional down-regulation of craA expression renders cells sensitive to chloramphenicol, a highly effective antibiotic for the treatment of multidrug resistant infections. An online interface that facilitates open data access and visualization is provided as 'AcinetoCom' (http://bioinf.gen.tcd.ie/acinetocom/).

Sinorhizobium meliloti is an alpha-proteobacterium able to induce nitrogen-fixing nodules on roots of specific legumes. In order to propagate in the soil and for successful symbiotic interaction the bacterium needs to sequester metals like iron and manganese from its environment. The metal uptake has to be in turn tightly regulated to avoid toxic effects. In this report we describe the characterization of a chromosomal region of S. meliloti encoding the sitABCD operon and the putative regulatory fur gene. It is generally assumed that the sitABCD operon encodes a metal-type transporter and that the fur gene is involved in iron ion uptake regulation. A constructed S. meliloti sitA deletion mutant was found to be growth dependent on Mn(II) and to a lesser degree on Fe(II). The sitA promoter was strongly repressed by Mn(II), with dependence on Fur, and moderately by Fe(II). Applying a genome-wide S. meliloti microarray it was shown that in the fur deletion mutant 23 genes were up-regulated and 10 genes were down-regulated when compared to the wild-type strain. Among the up-regulated genes only the sitABCD operon could be associated with metal uptake. On the other hand, the complete rhbABCDEF operon, which is involved in siderophore synthesis, was identified among the down-regulated genes. Thus, in S. meliloti Fur is not a global repressor of iron uptake. Under symbiotic conditions the sitA promoter was strongly expressed and the S. meliloti sitA mutant exhibited an attenuated nitrogen fixation activity resulting in a decreased fresh weight of the host plant Medicago sativa.