Homologous recombination between tandem paralogues drives evolution of Type VII secretion system immunity genes in firmicute bacteria

The Type VII secretion system (T7SS) is found in many Gram-positive firmicutes and secretes protein toxins that mediate bacterial antagonism. Two T7SS toxins have been identified in Staphylococcus aureus, EsaD a nuclease toxin that is counteracted by the EsaG immunity protein, and TspA, which has membrane depolarising activity and is neutralised by TsaI. Both toxins are polymorphic, and strings of non-identical esaG and tsaI immunity genes are encoded in all S. aureus strains. During genome sequence analysis of closely related S. aureus strains we noted that there had been a deletion of six consecutive esaG copies in one lineage. To investigate this further, we analysed the sequences of the tandem esaG genes and their encoded proteins. We identified three blocks of high sequence homology shared by all esaG genes, and identified evidence of extensive recombination events between esaG paralogues facilitated through these conserved sequence blocks. Recombination between these blocks accounts for loss of esaG genes from S. aureus genomes. TipC, an immunity protein for the TelC lipid II phosphatase toxin secreted by the streptococcal T7SS, is also encoded by multiple gene paralogues. Two blocks of high sequence homology locate to the 5-prime and 3-prime end of tipC genes, and we found strong evidence for recombination between tipC paralogues encoded by Streptococcus mitis BCC08. By contrast, we found only a single block of homology across tsaI genes, and little evidence for intergenic recombination within this gene family. We conclude that homologous recombination is one of the drivers for the evolution of T7SS immunity gene clusters.

Identification of candidate type 3 effectors that determine host specificity associated with emerging Ralstonia pseudosolanacearum strains

Ralstonia pseudosolanacearum (Rps), previously known as R. solanacearum phylotypes I and III is one of the causal agents of bacterial wilt, a devastating disease that affects more than 250 plant species. Emerging Rps strains were identified infecting new hosts. P824 Rps strain was isolated from blueberry in Florida. Rps strains including PD7123 were isolated from hybrid tea roses in several countries through Europe. P781 is a representative strain of Rps commonly found on mandevilla in Florida. UW757 is a strain isolated from osteospermum plants originating in Guatemala. These strains are phylogenetically closely related and of economic importance on their respective hosts. The objective of this study is to associate the Type 3 Effectors (T3Es) repertoire of these four strains with host specificity. Candidate T3E associated with host specificity to blueberry, tea rose, osteospermum, and mandevilla were identified by sequence homology. Pathogenicity assays on 8 hosts including, blueberry, mandevilla, osteospermum and tea rose with the 4 strains showed that both P824 and PD7123 are pathogenic to blueberry and tea rose. P781 is the only strain pathogenic to mandevilla and P824 is the only strain non-pathogenic to osteospermum. Hypotheses based on correlation of T3E presence/absence and pathogenicity profiles identified 3 candidate virulence and 3 avirulence T3E for host specificity to blueberry and tea rose. Two candidate avirulence T3E were identified for mandevilla, and one candidate virulence for osteospermum. The strategy applied here can be used to reduce the number of host specificity candidate genes in closely related strains with different hosts.

SLUPT: Synthesis of libraries and multi-site mutagenesis using a PCR-derived, dU-containing template Protocol

SLUPT stands for Synthesis of Libraries and Multi-Site Mutagenesis using a PCR derived, U-containing Template. SLUPT enables the user to create a DNA library, and/or multiple mutations of a given DNA target, independently or simultaneously. In particular, when SLUPT is used in conjunction with known structural and sequence homology data, one can create highly directed libraries/mutations spread across the gene of interest.

Structures of prokaryotic ubiquitin-like protein Pup in complex with depupylase Dop reveal the mechanism of catalytic phosphate formation

Pupylation is the post-translational modification of lysine side chains with prokaryotic ubiquitin-like protein (Pup) that targets proteins for proteasomal degradation in mycobacteria and other members of Actinobacteria. Pup ligase PafA and depupylase Dop are the two enzymes acting in this pathway. Although they share close structural and sequence homology indicative of a common evolutionary origin, they catalyze opposing reactions. Here, we report a series of high-resolution crystal structures of Dop in different functional states along the reaction pathway, including Pup-bound states in distinct conformations. In combination with biochemical analysis, the structures explain the role of the C-terminal residue of Pup in ATP hydrolysis, the process that generates the catalytic phosphate in the active site, and suggest a role for the Dop-loop as an allosteric sensor for Pup-binding and ATP cleavage.

Chinese experience in the management and control of COVID-19 epidemic

In December 2019, a new epidemic of coronavirus disease appeared (COVID–19), caused by SARS-CoV-2 (formerly 2019-nCoV). The first reported disease cases occurred in Wuhan, Hubei province, resulting in the third zoonotic event related to lethal human coronavirus. Initially, the incubation period is 1-14 days (mean 5-6 days) in most cases but can be as long as 24 days1,2. The most commonly seen characteristics of COVID-19 are fever, cough, tiredness, and abnormal chest computed tomography3,4. So far, bat is thought to be the origin of SARS-CoV-2, based on sequence homology of 96% between SARS-CoV-2 and Bat-CoV-RaTG135,6,7, but more impartial scientific investigations on the origin-tracing of the virus are required to elucidate the issue. Human-to-human transmission of SARS-CoV-2 occurs mainly via respiratory droplets1, direct contact1, asymptomatic transmission8,9 , and intrafamilial transmission3,4. At present, there are over 206 million cases of COVID-19 worldwide, with a 4.35 million death toll10. As of 12 August 2021, China had confirmed 94,260 cases with 4636 deaths (mortality rate 5%), and 87,740 recovered cases (93%)11. Other countries, even though they had much more time to prepare for the arrival of the virus, delayed their response and that meant lost control12. While the world is struggling to control COVID-19, China has been a good example of how to control the epidemic, and has shared information with other countries on the management and prevention of the disease. How was that possible?

A WDR35-dependent coat protein complex transports ciliary membrane cargo vesicles to cilia

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits, and demonstrate an accumulation of 'coat-less' vesicles which fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in-situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation.

Murine SEC24D can substitute functionally for SEC24C during embryonic development

The COPII component SEC24 mediates the recruitment of transmembrane cargos or cargo adaptors into newly forming COPII vesicles on the ER membrane. Mammalian genomes encode four Sec24 paralogs (Sec24a-d), with two subfamilies based on sequence homology (SEC24A/B and C/D), though little is known about their comparative functions and cargo-specificities. Complete deficiency for Sec24d results in very early embryonic lethality in mice (before the 8 cell stage), with later embryonic lethality (E7.5) observed in Sec24c null mice. To test the potential overlap in function between SEC24C/D, we employed dual recombinase mediated cassette exchange to generate a Sec24cc-d allele, in which the C-terminal 90% of SEC24C has been replaced by SEC24D coding sequence. In contrast to the embryonic lethality at E7.5 of SEC24C-deficiency, Sec24cc-d/c-d pups survive to term, though dying shortly after birth. Sec24cc-d/c-d pups are smaller in size, but exhibit no other obvious developmental abnormality by pathologic evaluation. These results suggest that tissue-specific and/or stage-specific expression of the Sec24c/d genes rather than differences in cargo export function explain the early embryonic requirements for SEC24C and SEC24D.

Molecular cloning and characterization of NAD+ dependent isocitrate dehydrogenase enzyme from Shewanella putrefaciens

Isocitrate dehydrogenase (IDH) is a fundamental enzyme for carbon metabolism in the Krebs cycle. This enzyme is required for oxidation-reduction reactions in both eukaryotic and prokaryotic cells and plays a critical role in their growth and pathogenesis. In this study, we cloned the gene encoding NAD+ dependent isocitrate dehydrogenase from Shewanella putrefaciens. The expression of recombinant protein was induced with 0.5 mM of IPTG. His-tagged IDH overexpressed in E. coli was purified and characterized. The expressed IDH enzyme was purified in an active soluble form. The molecular weight of the enzyme was confirmed with Western blotting. High sequence homology was observed with IDH sequences of other Shewanella strains and remarkable sequence homology was found with other bacteria reported in the database. The open reading frame of the gene encoding IDH of S. putrefaciens was 1069 bp in length, which codes a polypeptide composed of 356 amino acids and with a 38 KDa molecular weight. The optimum enzyme activity was obtained at pH 8. We cloned, purified and characterized Shewanella putrefaciens isocitrate dehydrogenase enzyme (SpIDH). The recombinant enzyme demonstrated a specific activity of Vmax 4.6±0.3 μM/min and Km 334 μM using NAD+ as a cofactor.