Substrate translocation involves specific lysine residues of the central channel of the conjugative coupling protein TrwB

Conjugative transfer of plasmid R388 requires the coupling protein TrwB for protein and DNA transport, but their molecular role in transport has not been deciphered. We investigated the role of residues protruding into the central channel of the TrwB hexamer by a mutational analysis. Mutations affecting lysine residues K275, K398, and K421, and residue S441, all facing the internal channel, affected transport of both DNA and the relaxase protein in vivo. The ATPase activity of the purified soluble variants was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour in vivo. Alteration of residues located at the cytoplasmic or the inner membrane interface resulted in lower activity in vivo and in vitro, while variants affecting residues in the central region of the channel showed increased DNA and protein transfer efficiency and higher ATPase activity, especially in the absence of TrwA. In fact, these variants could catalyze DNA transfer in the absence of TrwA under conditions in which the wild-type system was transfer deficient. Our results suggest that protein and DNA molecules have the same molecular requirements for translocation by Type IV secretion systems, with residues at both ends of the TrwB channel controlling the opening?closing mechanism, while residues embedded in the channel would set the pace for substrate translocation (both protein and DNA) in concert with TrwA

The Effect of Iron Limitation on the Transcriptome and Proteome of Pseudomonas fluorescens Pf-5

One of the most important micronutrients for bacterial growth is iron, whose bioavailability in soil is limited. Consequently, rhizospheric bacteria such as Pseudomonas fluorescens employ a range of mechanisms to acquire or compete for iron. We investigated the transcriptomic and proteomic effects of iron limitation on P. fluorescens Pf-5 by employing microarray and iTRAQ techniques, respectively. Analysis of this data revealed that genes encoding functions related to iron homeostasis, including pyoverdine and enantio-pyochelin biosynthesis, a number of TonB-dependent receptor systems, as well as some inner-membrane transporters, were significantly up-regulated in response to iron limitation. Transcription of a ribosomal protein L36-encoding gene was also highly up-regulated during iron limitation. Certain genes or proteins involved in biosynthesis of secondary metabolites such as 2,4-diacetylphloroglucinol (DAPG), orfamide A and pyrrolnitrin, as well as a chitinase, were over-expressed under iron-limited conditions. In contrast, we observed that expression of genes involved in hydrogen cyanide production and flagellar biosynthesis were down-regulated in an iron-depleted culture medium. Phenotypic tests revealed that Pf-5 had reduced swarming motility on semi-solid agar in response to iron limitation. Comparison of the transcriptomic data with the proteomic data suggested that iron acquisition is regulated at both the transcriptional and post-transcriptional levels

Huanglongbing alters the structure and functional diversity of microbial communities associated with citrus rhizosphere

The diversity and stability of bacterial communities present in the rhizosphere heavily influence soil and plant quality and ecosystem sustainability. The goal of this study is to understand how ‘Candidatus Liberibacter asiaticus' (known to cause Huanglongbing, HLB) influences the structure and functional potential of microbial communities associated with the citrus rhizosphere. Clone library sequencing and taxon/group-specific quantitative real-time PCR results showed that ‘Ca. L. asiaticus' infection restructured the native microbial community associated with citrus rhizosphere. Within the bacterial community, phylum Proteobacteria with various genera typically known as successful rhizosphere colonizers were significantly greater in clone libraries from healthy samples, whereas phylum Acidobacteria, Actinobacteria and Firmicutes, typically more dominant in the bulk soil were higher in ‘Ca. L. asiaticus'-infected samples. A comprehensive functional microarray GeoChip 3.0 was used to determine the effects of ‘Ca. L. asiaticus' infection on the functional diversity of rhizosphere microbial communities. GeoChip analysis showed that HLB disease has significant effects on various functional guilds of bacteria. Many genes involved in key ecological processes such as nitrogen cycling, carbon fixation, phosphorus utilization, metal homeostasis and resistance were significantly greater in healthy than in the ‘Ca. L. asiaticus'-infected citrus rhizosphere. Our results showed that the microbial community of the ‘Ca. L. asiaticus'-infected citrus rhizosphere has shifted away from using more easily degraded sources of carbon to the more recalcitrant forms. Overall, our study provides evidence that the change in plant physiology mediated by ‘Ca. L. asiaticus' infection could elicit shifts in the composition and functional potential of rhizosphere microbial communities. In the long term, these fluctuations might have important implications for the productivity and sustainability of citrus-producing agro-ecosystems

DNA damage emergency: cellular garbage disposal to the rescue?

The proteasome is a cellular machine found in the cytosol, nucleus and on chromatin that performs much of the proteolysis in eukaryotic cells. Recent reports show it is enriched at sites of double-stranded DNA breaks (DSBs) in mammalian cells. What is it doing there? This review will address three possibilities suggested by recent reports: in degrading proteins after their ubiquitination at and eviction from chromatin; as a deubiquitinase, specific to the antagonism of ubiquitin conjugates generated as part of the signalling of a DSB; and as a functional component of DNA repair mechanism itself. These findings add complexity to the proteasome as a potential therapeutic target in cancer treatment