BACKGROUND: Autolysis by cellular peptidoglycan hydrolases (PGH) is a well-known phenomenon in bacteria. During food fermentation, autolysis of starter cultures can exert an accelerating effect, as described in many studies on cheese ripening. In contrast, very little is known about autolysis of starter cultures used in other fermentations. Staphylococcus (S.) carnosus is often used in raw sausage fermentations, contributing to nitrate reduction and flavor formation. In this study, we analyzed the influence of PGHs of the strains S. carnosus TMW 2.146 and S. carnosus TMW 2.2525 on their autolytic behavior. The staphylococcal major autolysin (Atl), a bifunctional enzyme with an N-acetylmuramoyl-L-alanine amidase and a glucosaminidase as an active site, is assumed to be the enzyme by which autolysis is mainly mediated.
RESULTS: AtlC mutant strains showed impaired growth and almost no autolysis compared to their respective wild-type strains. Light microscopy and scanning electron microscopy showed that the mutants could no longer appropriately separate from each other during cell division, resulting in the formation of cell clusters. The surface of the mutants appeared rough with an irregular morphology compared to the smooth cell surfaces of the wild-types. Moreover, zymograms showed that eight lytic bands of S. carnosus, with a molecular mass between 140 and 35 kDa, are processed intermediates of AtlC. It was noticed that additional bands were found that had not been described in detail before and that the banding pattern changes over time. Some bands disappear entirely, while others become stronger or are newly formed. This suggests that AtlC is degraded into smaller fragments over time. A second knockout was generated for the gene encoding a N-acetylmuramoyl-L-alanine amidase domain-containing protein. Still, no phenotypic differences could be detected in this mutant compared to the wild-type, implying that the autolytic activity of S. carnosus is mediated by AtlC.
CONCLUSIONS: In this study, two knockout mutants of S. carnosus were generated. The atlC mutant showed a significantly altered phenotype compared to the wild-type, revealing AtlC as a key factor in staphylococcal autolysis. Furthermore, we show that Atl is degraded into smaller fragments, which are still cell wall lytic active.

Rice yellow mottle virus (RYMV) causes one of the most devastating rice diseases in Africa. Management of RYMV is challenging. Genetic resistance provides the most effective and environment-friendly control. The recessive resistance locus rymv2 (OsCPR5.1) had been identified in African rice (Oryza glaberrima), however, introgression into Oryza sativa ssp. japonica and indica remains challenging due to crossing barriers. Here, we evaluated whether CRISPR/Cas9 genome editing of the two rice nucleoporin paralogs OsCPR5.1 (RYMV2) and OsCPR5.2 can be used to introduce RYMV resistance into the japonica variety Kitaake. Both paralogs had been shown to complement the defects of the Arabidopsis atcpr5 mutant, indicating partial redundancy. Despite striking sequence and structural similarities between the two paralogs, only oscpr5.1 loss-of-function mutants were fully resistant, while loss-of-function oscpr5.2 mutants remained susceptible, intimating that OsCPR5.1 plays a specific role in RYMV susceptibility. Notably, edited lines with short in-frame deletions or replacements in the N-terminal domain (predicted to be unstructured) of OsCPR5.1 were hypersusceptible to RYMV. In contrast to mutations in the single Arabidopsis AtCPR5 gene, which caused severely dwarfed plants, oscpr5.1 and oscpr5.2 single and double knockout mutants showed neither substantial growth defects nor symptoms indicative lesion mimic phenotypes, possibly reflecting functional differentiation. The specific editing of OsCPR5.1, while maintaining OsCPR5.2 activity, provides a promising strategy for generating RYMV-resistance in elite Oryza sativa lines as well as for effective stacking with other RYMV resistance genes or other traits.

Nocardia seriolae is the main pathogen of fish nocardiosis. In our previous study, alanine dehydrogenase was identified as a potential virulence factor of N. seriolae. On the basis of this fact, the alanine dehydrogenase gene of N. seriolae (NsAld) was knocked out to establish the strain ΔNsAld for vaccine development against fish nocardiosis in this study. The LD50 of strain ΔNsAld was 3.90 × 105 CFU/fish, higher than that of wild strain (5.28 × 104 CFU/fish) significantly (p < 0.05). When the strain ΔNsAld was used as a live vaccine to immunize hybrid snakehead (Channa maculata ♀ × Channa argus ♂) at 2.47 × 105 CFU/fish by intraperitoneal injection, the non-specific immune indexes (LZM, CAT, AKP, ACP and SOD activities), specific antibody (IgM) titers and several immune-related genes (CD4, CD8α, IL-1β, MHCIα, MHCIIα and TNFα) were up-regulated in different tissues, indicating that this vaccine could induce humoral and cell-mediated immune responses. Furthermore, the relative percentage survival (RPS) of ΔNsAld vaccine was calculated as 76.48% after wild N. seriolae challenge. All these results suggest that the strain ΔNsAld could be a potential candidate for live vaccine development to control fish nocardiosis in aquaculture.

Chromoblastomycosis (CBM) is a disease caused by several dematiaceous fungi from different genera, and Fonsecaea is the most common which has been clinically isolated. Genetic transformation methods have recently been described; however, molecular tools for the functional study of genes have been scarcely reported for those fungi. In this work, we demonstrated that gene deletion and generation of the null mutant by homologous recombination are achievable for Fonsecaea pedrosoi by the use of two approaches: use of double-joint PCR for cassette construction, followed by delivery of the split-marker by biolistic transformation. Through in silico analyses, we identified that F. pedrosoi presents the complete enzymatic apparatus required for tryptophan (trp) biosynthesis. The gene encoding a tryptophan synthase trpB -which converts chorismate to trp-was disrupted. The ΔtrpB auxotrophic mutant can grow with external trp supply, but germination, viability of conidia, and radial growth are defective compared to the wild-type and reconstituted strains. The use of 5-FAA for selection of trp- phenotypes and for counter-selection of strains carrying the trp gene was also demonstrated. The molecular tools for the functional study of genes, allied to the genetic information from genomic databases, significantly boost our understanding of the biology and pathogenicity of CBM causative agents.

L-2-halocid dehalogenases (L-2-HADs) have been mainly characterized from terrestrial polluted environments. By contrast, knowledge is still scarce about their role in detoxification of predominant halocarbons in marine environments. Here, phylogenetic analyses showed a wide diversity of homologous L-2-HADs, especially among those belonging to marine bacteria. Previously characterized terrestrial L-2-HADs were part of a monophyletic group (named group A) including proteins of terrestrial and marine origin. Another branch (named group B) contained mostly marine L-2-HADs, with two distinct clades of Bacteroidetes homologs, closely linked to Proteobacteria ones. This study further focused on the characterization of the only L-2-HAD from the flavobacterium Zobellia galactanivorans DsijT (ZgHAD), belonging to one of these Group B clades. The recombinant ZgHAD was shown to dehalogenate bromo- and iodoacetic acids, and gene knockout in Z. galactanivorans revealed a direct role of ZgHAD in tolerance against both haloacetic acids. Analyses of metagenomic and metatranscriptomic datasets confirmed that L-2-HADs from group A were well-represented in terrestrial and marine bacteria, whereas ZgHAD homologs (group B L-2-HADs) were mainly present in marine bacteria, and particularly in host-associated species. Our results suggest that ZgHAD homologs could be key enzymes for marine Bacteroidetes, by conferring selective advantage for the recycling of toxic halogen compounds produced in particular marine habitats, and especially during interactions with macroalgae.

Pamamycins, a group of polyketides originally discovered in Streptomyces alboniger, induce sporulation in Streptomyces and inhibit the growth of Gram-positive bacteria, Mycobacterium tuberculosis and fungi. The pamamycin biosynthetic gene cluster encodes 6 ketosynthases that utilize a variety of three-carbon to five-carbon CoA thioesters as starter and extender units. This promiscuity in production results in an up to 18 different derivatives during fermentation. For more-selective production and simplified purification, we aimed to modify the precursor supply to narrow the spectrum of the produced derivatives. Eight genes potentially responsible for the supply of two major precursors, 2-S-methylmalonyl-CoA and 2-S-ethylmalonyl-CoA, were identified using the NCBI Basic Local Alignment Search Tool (BLAST) against the genome of the heterologous host S. albus J1074. Knockout mutants of the identified genes were constructed and their impact on intracellular CoA ester concentrations and on the production of pamamycins was determined. The created mutants enabled us to conclusively identify the ethylmalonyl-CoA supplying routes and their impact on the production of pamamycin. Furthermore, we gained significant information on the origin of the methylmalonyl-CoA supply in Streptomyces albus.

Penicillium digitatum is the main fungal postharvest pathogen of citrus fruit under Mediterranean climate conditions. The role of ethylene in the P. digitatum-citrus fruit interaction is unclear and controversial. We analyzed the involvement of the 2-oxoglutarate-dependent ethylene-forming enzyme (EFE)-encoding gene (efeA) of P. digitatum on the pathogenicity of the fungus. The expression of P. digitatumefeA parallels ethylene production during growth on PDA medium, with maximum levels reached during sporulation. We generated ΔefeA knockout mutants in P. digitatum strain Pd1. These mutants showed no significant defect on mycelial growth or sporulation compared to the parental strain. However, the knockout mutants did not produce ethylene in vitro. Citrus pathogenicity assays showed no differences in virulence between the parental and ΔefeA knockout mutant strains, despite a lack of ethylene production by the knockout mutant throughout the infection process. This result suggests that ethylene plays no role in P. digitatum pathogenicity. Our results clearly show that EFE-mediated ethylene synthesis is the major ethylene synthesis pathway in the citrus postharvest pathogen P. digitatum during both in vitro growth on PDA medium and the infection process, and that this hormone is not necessary for establishing P. digitatum infection in citrus fruit. However, our results also indicate that ethylene produced by P. digitatum during sporulation on the fruit surface may influence the development of secondary fungal infections.

The MAPEG2 sub-family of glutathione-S-transferase proteins (GST) has been poorly investigated in vivo, even in prokaryotes such as cyanobacteria the organisms that are regarded as having developed glutathione-dependent enzymes to protect themselves against the reactive oxygen species (ROS) often produced by their powerful photosynthesis. We report the first in vivo analysis of a cyanobacterial MAPEG2-like protein (Sll1147) in the model cyanobacterium Synechocystis PCC 6803. While Sll1147 is dispensable to cell growth in standard photo-autotrophic conditions, it plays an important role in the resistance to heat and cold, and to n-tertbutyl hydroperoxide (n-tBOOH) that induces lipid peroxidation. These findings suggest that Sll1147 could be involved in membrane fluidity, which is critical for photosynthesis. Attesting its sensitivity to these stresses, the Δsll1147 mutant lacking Sll1147 challenged by heat, cold, or n-tBOOH undergoes transient accumulation of peroxidized lipids and then of reduced and oxidized glutathione. These results are welcome because little is known concerning the signaling and/or protection mechanisms used by cyanobacteria to cope with heat and cold, two inevitable environmental stresses that limit their growth, and thus their production of biomass for our food chain and of biotechnologically interesting chemicals. Also interestingly, the decreased resistance to heat, cold and n-tBOOH of the Δsll1147 mutant could be rescued back to normal (wild-type) levels upon the expression of synthetic MAPEG2-encoding human genes adapted to the cyanobacterial codon usage. These synthetic hmGST2 and hmGST3 genes were also able to increase the Escherichia coli tolerance to heat and n-tBOOH. Collectively, these finding indicate that the activity of the MAPEG2 proteins have been conserved, at least in part, during evolution from (cyano)bacteria to human.

The effect of central metabolic activity of Escherichia coli cells acting as biocatalysts on the performance of microbial fuel cells (MFCs) was studied with glucose used as the energy source. Milliliter-scale two-chambered MFCs were used with 2-hydroxy-1,4-naphthoquinone (HNQ) as an electron mediator. Among the single-gene deletions examined, frdA, pdhR, ldhA, and adhE increased the average power output of the constructed MFC. Next, multiple-gene knockout mutants were constructed using P1 transduction. The Δ5 (ΔfrdAΔpdhRΔldhAΔadhEΔpta) strain showed the highest ave. power output (1.82 mW) and coulombic efficiency (21.3%). Our results show that the combination of multiple-gene knockout in E. coli cells leads to the development of an excellent catalyst for MFCs. Finally, preventing a decrease in the pH of the anodic solution was a key factor for improving the power output of the Δ5 strain, and a maximum ave. power output of 2.21 mW was achieved with 5% NaHCO3 in the buffer. The ave. power density of the constructed MFC was 0.27 mW/cm3, which is comparable to an enzymatic fuel cell of a Milliliter-scale using glucose dehydrogenase.

Persisters comprise a group of phenotypically heterogeneous metabolically quiescent bacteria with multidrug tolerance and contribute to the recalcitrance of chronic infections. Although recent work has shown that toxin-antitoxin (TA) system HipAB depends on stringent response effector (p)ppGppin persister formation, whether other persister pathways are also dependent on stringent response has not been explored. Here we examined the relationship of (p)ppGpp with 15 common persister genes (dnaK, clpB, rpoS, pspF, tnaA, sucB, ssrA, smpB, recA, umuD, uvrA, hipA, mqsR, relE, dinJ) using Escherichia coli as a model. By comparing the persister levels of wild type with their single gene knockout and double knockout mutants with relA, we divided their interactions into five types, namely A \"dependent\" (dnaK, recA), B \"positive reinforcement\" (rpoS, pspF, ssrA, recA), C \"antagonistic\" (clpB, sucB, umuD, uvrA, hipA, mqsR, relE, dinJ), D \"epistasis\" (clpB, rpoS, tnaA, ssrA, smpB, hipA), and E \"irrelevant\" (dnaK, clpB, rpoS, tnaA, sucB, smpB, umuD, uvrA, hipA, mqsR, relE, dinJ). We found that the persister gene interactions are intimately dependent on bacterial culture age, cell concentrations (diluted versus undiluted culture), and drug classifications, where the same gene may belong to different groups with varying antibiotics, culture age or cell concentrations. Together, this study represents the first attempt to systematically characterize the intricate relationships among the different mechanisms of persistence and as such provide new insights into the complexity of the persistence phenomenon at the level of persister gene network interactions.