Comprehensive Proteome Profiling of a Xanthomonas campestris pv. Campestris B100 Culture Grown in Minimal Medium with a Specific Focus on Nutrient Consumption and Xanthan Biosynthesis.

Xanthan, a bacterial polysaccharide, is widespread in industrial applications, particularly as a food additive. However, little is known about the process of xanthan synthesis on the proteome level, even though Xanthomonas campestris is frequently used for xanthan fermentation. A label-free LC-MS/MS method was employed to study the protein changes during xanthan fermentation in minimal medium. According to the reference database, 2416 proteins were identified, representing 54.75 % of the proteome. The study examined changes in protein abundances concerning the growth phase and xanthan productivity. Throughout the experiment, changes in nitrate concentration appeared to affect the abundance of most proteins involved in nitrogen metabolism, except Gdh and GlnA. Proteins involved in sugar nucleotide metabolism stay unchanged across all growth phases. Apart from GumD, GumB, and GumC, the gum proteins showed no significant changes throughout the experiment. GumD, the first enzyme in the assembly of the xanthan-repeating unit, peaked during the early stationary phase but decreased during the late stationary phase. GumB and GumC, which are involved in exporting xanthan, increased significantly during the stationary phase. This study suggests that a potential bottleneck for xanthan productivity does not reside in the abundance of proteins directly involved in the synthesis pathways.

Systems biology of industrial oxytetracycline production in Streptomyces rimosus: the secrets of a mutagenized hyperproducer.

BACKGROUND: Oxytetracycline which is derived from Streptomyces rimosus, inhibits a wide range of bacteria and is industrially important. The underlying biosynthetic processes are complex and hinder rational engineering, so industrial manufacturing currently relies on classical mutants for production. While the biochemistry underlying oxytetracycline synthesis is known to involve polyketide synthase, hyperproducing strains of S. rimosus have not been extensively studied, limiting our knowledge on fundamental mechanisms that drive production. RESULTS: In this study, a multiomics analysis of S. rimosus is performed and wild-type and hyperproducing strains are compared. Insights into the metabolic and regulatory networks driving oxytetracycline formation were obtained. The overproducer exhibited increased acetyl-CoA and malonyl CoA supply, upregulated oxytetracycline biosynthesis, reduced competing byproduct formation, and streamlined morphology. These features were used to synthesize bhimamycin, an antibiotic, and a novel microbial chassis strain was created. A cluster deletion derivative showed enhanced bhimamycin production. CONCLUSIONS: This study suggests that the precursor supply should be globally increased to further increase the expression of the oxytetracycline cluster while maintaining the natural cluster sequence. The mutagenized hyperproducer S. rimosus HP126 exhibited numerous mutations, including large genomic rearrangements, due to natural genetic instability, and single nucleotide changes. More complex mutations were found than those typically observed in mutagenized bacteria, impacting gene expression, and complicating rational engineering. Overall, the approach revealed key traits influencing oxytetracycline production in S. rimosus, suggesting that similar studies for other antibiotics could uncover general mechanisms to improve production.

RcgA and RcgR, Two Novel Proteins Involved in the Conjugative Transfer of Rhizobial Plasmids.

Rhizobia are Gram-negative bacteria that are able to establish a nitrogen-fixing symbiotic interaction with leguminous plants. Rhizobia genomes usually harbor several plasmids which can be transferred to other organisms by conjugation. Two main mechanisms of the regulation of rhizobial plasmid transfer have been described: quorum sensing (QS) and the rctA/rctB system. Nevertheless, new genes and molecules that modulate conjugative transfer have recently been described, demonstrating that new actors can tightly regulate the process. In this work, by means of bioinformatics tools and molecular biology approaches, two hypothetical genes are identified as playing key roles in conjugative transfer. These genes are located between conjugative genes of plasmid pRfaLPU83a from Rhizobium favelukesii LPU83, a plasmid that shows a conjugative transfer behavior depending on the genomic background. One of the two mentioned genes, rcgA, is essential for conjugation, while the other, rcgR, acts as an inhibitor of the process. In addition to introducing this new regulatory system, we show evidence of the functions of these genes in different genomic backgrounds and confirm that homologous proteins from non-closely related organisms have the same functions. These findings set up the basis for a new regulatory circuit of the conjugative transfer of plasmids. IMPORTANCE Extrachromosomal DNA elements, such as plasmids, allow for the adaptation of bacteria to new environments by conferring new determinants. Via conjugation, plasmids can be transferred between members of the same bacterial species, different species, or even to organisms belonging to a different kingdom. Knowledge about the regulatory systems of plasmid conjugative transfer is key in understanding the dynamics of their dissemination in the environment. As the increasing availability of genomes raises the number of predicted proteins with unknown functions, deeper experimental procedures help to elucidate the roles of these determinants. In this work, two uncharacterized proteins that constitute a new regulatory circuit with a key role in the conjugative transfer of rhizobial plasmids were discovered.