Our integrated approach, using a metabolic model in conjunction with proteomics measurements, enabled quantification of uncertainty across various pathway targets to improve the efficiency of isopropanol bioproduction. From in silico thermodynamic optimization, minimal protein requirement analysis, and ensemble modeling-based robustness analysis, acetoacetyl-coenzyme A (CoA) transferase (AACT) and acetoacetate decarboxylase (AADC) were identified as the prime flux control sites. Elevated isopropanol production is projected with the overexpression of these. Our predictions served as the blueprint for iterative pathway construction, resulting in a 28-fold increase in isopropanol production when contrasted with the initial version. Under gas-fermenting mixotrophic conditions, the engineered strain underwent additional testing. Carbon monoxide, carbon dioxide, and fructose were employed as substrates, resulting in isopropanol production exceeding 4 grams per liter. CO2, CO, and H2 sparging in a bioreactor environment yielded 24 g/L isopropanol production by the strain. Directed and intricate pathway engineering has been shown by our work to be a critical element for achieving high-yield bioproduction using gas-fermenting chassis. A crucial aspect of highly efficient bioproduction from gaseous substrates (hydrogen and carbon oxides) is the systematic optimization of the host microbial communities. So far, the rational redesign of gas-fermenting bacteria is still underdeveloped, largely because of the absence of accurate and detailed metabolic data required to effectively guide strain engineering. The presented case study highlights the engineering challenges and solutions for the production of isopropanol by the gas-fermenting Clostridium ljungdahlii. We show how a modeling strategy, built upon thermodynamic and kinetic pathway analyses, can yield practical knowledge for strain engineering, leading to optimal bioproduction. This approach potentially unlocks the path for iterative microbe redesign, facilitating the conversion of renewable gaseous feedstocks.

A major concern for human health is the emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP), whose proliferation is primarily attributed to a few dominant lineages, defined by their sequence types (ST) and capsular (KL) types. Among the dominant lineages, ST11-KL64 is particularly prevalent in China, as well as globally. The population structure and the provenance of ST11-KL64 K. pneumoniae are still subjects of ongoing research. The NCBI repository provided us with all K. pneumoniae genomes (13625, as of June 2022), comprising 730 strains, a specific type designated as ST11-KL64. Through phylogenomic analysis of the core genome, marked by single-nucleotide polymorphisms, two prominent clades (I and II) emerged, in addition to an isolated strain ST11-KL64. Our analysis of dated ancestral reconstruction, achieved using BactDating, indicated clade I's probable origination in Brazil in 1989, and clade II's probable origin in eastern China around 2008. Our subsequent inquiry into the origin of the two clades and the singleton involved a phylogenomic approach that also included the analysis of recombination regions. We observed a likely hybrid composition in the ST11-KL64 clade I, with an approximated 912% (approximately) contribution from a distinct ancestral line. The chromosome comprises 498Mb (88%) of genetic material from the ST11-KL15 lineage, and 483kb of genetic material sourced from the ST147-KL64 lineage. ST11-KL64 clade II, in contrast to ST11-KL47, is derived by the swapping of a 157 kb segment (approximately 3% of the chromosome), containing the capsule gene cluster, with the clonal complex 1764 (CC1764)-KL64 strain. From ST11-KL47, the singleton emerged, but its development was marked by an exchange of a 126-kb region with the ST11-KL64 clade I. Ultimately, ST11-KL64 represents a heterogeneous lineage, divided into two primary clades and an isolated branch, each originating in distinct countries and at various chronological points. Carbapenem-resistant Klebsiella pneumoniae (CRKP), a significant global threat, is strongly linked to increased hospital stays and high mortality in affected patients. A few predominant lineages, including ST11-KL64, a dominant strain in China, play a substantial role in the spread of CRKP globally. To determine if ST11-KL64 K. pneumoniae is a single genomic lineage, we carried out a genome-focused research project. Despite expectations, ST11-KL64's structure comprised a singleton and two large clades, independently arising in distinct countries and years. The two clades and the isolated lineage exhibit divergent evolutionary histories, having each acquired the KL64 capsule gene cluster from different ancestral sources. Tradipitant Our study reveals that the capsule gene cluster's chromosomal location is a prominent site of recombination in the K. pneumoniae bacterium. This evolutionary mechanism, crucial for rapid adaptation, is employed by certain bacteria to generate novel clades, enabling survival in stressful conditions.

The varied and antigenically distinct capsule types that Streptococcus pneumoniae can produce greatly hinder the effectiveness of vaccines targeting the pneumococcal polysaccharide (PS) capsule. Nevertheless, numerous pneumococcal capsule types continue to elude discovery and/or characterization. Analysis of pneumococcal capsule synthesis (cps) loci in prior sequences indicated the presence of capsule subtypes within isolates conventionally classified as serotype 36. Through our investigation, we found these subtypes to be two pneumococcal capsule serotypes, 36A and 36B, displaying comparable antigenicity but showing distinct characteristics. Biochemical investigation of the capsule PS structures in both cases reveals a shared repeat unit backbone, [5),d-Galf-(11)-d-Rib-ol-(5P6),d-ManpNAc-(14),d-Glcp-(1)], with two branch points. Ribitol is the endpoint of the -d-Galp branch present in both serotypes. Tradipitant Serotype 36A is characterized by a -d-Glcp-(13),d-ManpNAc branch, while serotype 36B contains a -d-Galp-(13),d-ManpNAc branch. Differences in the incorporation of Glcp (in serogroups 9N and 36A) versus Galp (in serogroups 9A, 9V, 9L, and 36B) were observed when comparing the phylogenetically distant serogroup 9 and 36 cps loci, all encoding the same glycosidic bond. This difference is reflected in four differing amino acids of the cps-encoded glycosyltransferase WcjA. Improving the accuracy and reliability of sequencing-based capsule typing and the discovery of novel, serologically indistinguishable capsule variants depend on identifying the functional determinants of cps-encoded enzymes and how these affect capsular polysaccharide structure.

The outer membrane of Gram-negative bacteria receives lipoproteins through the action of the localization (Lol) system. Models of lipoprotein transfer by Lol proteins across the inner and outer membranes in Escherichia coli have been extensively characterized, but lipoprotein synthesis and export pathways in numerous bacterial species exhibit significant variations from the E. coli model. The E. coli outer membrane protein LolB has no counterpart in the human gastric bacterium Helicobacter pylori; the E. coli proteins LolC and LolE are functionally represented by the single inner membrane protein LolF; and the E. coli cytoplasmic ATPase LolD is not identified in this organism. We undertook this present study to identify a protein similar to LolD in the context of H. pylori. Tradipitant Employing affinity-purification and mass spectrometry, we determined the interaction partners of the H. pylori ATP-binding cassette (ABC) family permease LolF. The identification of HP0179, an ABC family ATP-binding protein, as an interaction partner is a key finding. We developed H. pylori strains that conditionally express HP0179, demonstrating that HP0179, along with its conserved ATP-binding and ATPase domains, are critical for the growth of H. pylori. Our affinity purification-mass spectrometry procedure, utilizing HP0179 as the bait, yielded the identification of LolF as a binding partner. H. pylori HP0179's classification as a LolD-like protein underscores our improved comprehension of lipoprotein localization procedures within H. pylori, a bacterium in which the Lol system presents a departure from the E. coli standard. Lipoproteins in Gram-negative bacteria are critical for the arrangement of LPS on the cellular surface, the integration of outer membrane proteins, and the recognition of envelope stress signals. Bacterial pathogenesis is further influenced by the presence of lipoproteins. The Gram-negative outer membrane is essential for the proper localization of lipoproteins in many of these functions. By way of the Lol sorting pathway, lipoproteins are transported to the outer membrane. Extensive studies of the Lol pathway have been undertaken in the model organism Escherichia coli, however, numerous bacteria employ alternative components or lack essential components that are present in the E. coli Lol pathway. For a more complete understanding of the Lol pathway in many bacterial groups, the discovery of a LolD-like protein in Helicobacter pylori is a significant step. Antimicrobial development initiatives increasingly focus on the localization of lipoproteins.

Recent advances in human microbiome research have discovered the significant presence of oral microbes in the stools of patients suffering from dysbiosis. Still, the extent to which these invasive oral microorganisms might interact with the host's commensal intestinal microbiota and the effects on the host are not fully elucidated. In this proof-of-concept study, a novel model of oral-to-gut invasion was presented, using an in vitro model (M-ARCOL) replicating the human colon's physicochemical and microbial properties (lumen and mucus-associated microbes), a salivary enrichment technique, and whole-metagenome sequencing. An in vitro colon model, seeded with a fecal sample from a healthy adult, experienced an injection of enriched saliva from the same donor, simulating the oral invasion of the intestinal microbiota.