Applied and Environmental Microbiology最新文献

Postdocs are essential to microbial science and STEM academic workforces but are underpaid and receive little-to-no relocation benefits. PhDs foregoing postdoctoral training for lucrative industry and government jobs exit the academic pipeline, which imperils current scholarship and the future professoriate. Relocation to postdoc jobs is expensive, especially for recent graduates and international scholars, but academia rarely provides support. Solving this short-term liquidity pressure can increase productivity, job satisfaction, and the likelihood they remain in academia.

Ecological processes greatly shape microbial community assembly, but the driving factors remain unclear. Here, we compiled a metagenomic data set of microbial communities from global acid mine drainage (AMD) and explored the ecological features of microbial community linked to stochastic and deterministic processes from the perspective of species niche position, interaction patterns, gene functions, and viral infection. Our results showed that dispersal limitation (DL) (48.5%~93.5%) dominated the assembly of phylogenetic bin in AMD microbial community, followed by homogeneous selection (HoS) (3.1%~39.2%), heterogeneous selection (HeS) (1.4%~22.2%), and drift (DR) (0.2%~2.7%). The dominant process of dispersal limitation was significantly influenced by niche position in temperature (r = -0.518, P = 0.007) and dissolved oxygen (r = 0.471, P = 0.015). Network stability had a significantly negative correlation with the relative importance of dispersal limitation, while it had a positive correlation with selection processes, implying changes in network properties could be mediated by ecological processes. Furthermore, we found that ecological processes were mostly related to the gene functions of energy production and conversion (C), and amino acid transport and metabolism (E). Meanwhile, our results showed that the number of proviruses and viral genes involved in arsenic (As) resistance is negatively associated with the relative importance of ecological drift in phylogenetic bin assembly, implying viral infection might weaken ecological drift. Taken together, these results highlight that ecological processes are associated with ecological features at multiple levels, providing a novel insight into microbial community assembly in extremely acidic environments.

Importance: Unraveling the forces driving community assemblage is a core issue in microbial ecology, but how ecological constraints impose stochasticity and determinism remains unknown. This study presents a comprehensive investigation to uncover the association of ecological processes with species niche position, interaction patterns, microbial metabolisms, and viral infections, which provides novel insights into community assembly in extreme environments.

Over the past three decades, the integrase (Int) from Streptomyces phage phiC31 has become a valuable genome engineering tool across various species. phiC31 Int was thought to mediate unidirectional site-specific integration (attP × attB to attL and attR) in the absence of the phage-encoded recombination directionality factor (RDF). However, we have shown in this study that Int can also catalyze reverse excision (attL × attR to attP and attB) at low frequencies in Streptomyces lividans and Escherichia coli, causing genetic instability in engineered strains. To address this issue, we developed a two-plasmid co-conjugation (TPC) system. This system consists of an attP-containing integration vector and an Int expression suicide plasmid, both carrying oriT to facilitate efficient conjugation transfer from E. coli to Streptomyces. Using the TPC system, genetically stable integrants free of Int can be generated quickly and easily. The indigoidine-producing strains generated by the TPC system exhibited higher genetic stability and production efficiency compared to the indigoidine-producing strain generated by the conventional integration system, further demonstrating the utility of the TPC system in the field of biotechnology. We anticipate that the strategies presented here will be widely adopted for stable genetic engineering of industrial microbes using phage integrase-based integration systems.IMPORTANCELarge serine recombinases (LSRs), including the bacteriophage phiC31 integrase, were previously thought to allow only unidirectional site-specific integration (attP × attB to attL and attR). Our study is the first to show that the phiC31 integrase can also catalyze a low-efficiency reverse excision reaction in Streptomyces and E. coli without the involvement of the phage-encoded recombination directionality factor (RDF). The genetic instability caused by the low in vivo excisionase activity of the phiC31 integrase is a major challenge for biotechnological applications. Our study addresses this issue by developing a two-plasmid co-conjugation (TPC) system that facilitates the construction of Int-deficient genomic engineering strains. The Int-deficient integrants produced by this TPC system exhibit strong genetic stability for introduced genes and maintain stable production traits even in the absence of selection pressure, making them highly valuable for industrial applications.

During the establishment of the symbiosis with legume plants, rhizobia are exposed to hostile physical and chemical microenvironments to which adaptations are required. Stress response proteins including small heat shock proteins (sHSPs) were previously shown to be differentially regulated in bacteroids induced by Rhizobium leguminosarum bv. viciae UPM791 in different hosts. In this work, we undertook a functional analysis of the host-dependent sHSP RLV_1399. A rlv_1399-deleted mutant strain was impaired in the symbiotic performance with peas but not with lentil plants. Expression of rlv_1399 gene was induced under microaerobic conditions in a FnrN-dependent manner consistent with the presence of an anaerobox in its regulatory region. Overexpression of this sHSP improves the viability of bacterial cultures following exposure to hydrogen peroxide and to cationic nodule-specific cysteine-rich (NCR) antimicrobial peptides. Co-purification experiments have identified proteins related to nitrogenase synthesis, stress response, carbon and nitrogen metabolism, and to other relevant cellular functions as potential substrates for RLV_1399 in pea bacteroids. These results, along with the presence of unusually high number of copies of shsp genes in rhizobial genomes, indicate that sHSPs might play a relevant role in the adaptation of the bacteria against stress conditions inside their host.IMPORTANCEThe identification and analysis of the mechanisms involved in host-dependent bacterial stress response is important to develop optimal Rhizobium/legume combinations to maximize nitrogen fixation for inoculant development and might have also applications to extend nitrogen fixation to other crops. The data presented in this work indicate that sHSP RLV_1399 is part of the bacterial stress response to face specific stress conditions offered by each legume host. The identification of a wide diversity of sHSP potential targets reveals the potential of this protein to protect essential bacteroid functions. The finding that nitrogenase is the most abundant RLV_1399 substrate suggests that this protein is required to obtain an optimal nitrogen-fixing symbiosis.

This study explored the genomic alterations in Yarrowia lipolytica, a key yeast in industrial biotechnology, under both spontaneous and mutagen-induced conditions. Our findings reveal that spontaneous mutations occur at a rate of approximately 4 × 10^-10 events per base pair per cell division, primarily manifesting as single-nucleotide variations (SNVs) and small insertions and deletions (InDels). Notably, C-to-T/G-to-A transitions and C-to-A/G-to-T transversions dominate the spontaneous SNVs, while 1 bp deletions, likely resulting from template slippage, are the most frequent InDels. Furthermore, chromosomal aneuploidy and rearrangements occur, albeit at a lower frequency. Exposure to ultraviolet (UV) light, methylmethane sulfonate (MMS), and Zeocin significantly enhances the rates of SNVs and alters their mutational spectra in distinct patterns. Notably, Zeocin-induced SNVs are predominantly T-to-A and T-to-G substitutions, often occurring within the 5'-TGT^*-3' motif (^* denotes the mutated base). Additionally, Zeocin exhibits a higher potency in stimulating InDels compared to UV and MMS. Translesion DNA synthesis is implicated as the primary mechanism behind most Zeocin-induced SNVs and some InDels, whereas non-homologous end joining serves as the main pathway for Zeocin-mediated InDels. Intriguingly, the study identifies the gene YALI1_E21053g, encoding a protein kinase, as negatively associated with Zeocin resistance. Overall, our results not only deepened our knowledge about the genome evolution in Y. lipolytica but also provided reference to develop innovative strategies to harness its genetic potential.IMPORTANCEYarrowia lipolytica exhibits high environmental stress tolerance and lipid metabolism capabilities, making it a microorganism with significant industrial application potential. In this study, we investigated the genomic variation and evolutionary patterns of this yeast under both spontaneous and induced mutation conditions. Our results reveal distinctive mutation spectra induced by different mutagenic conditions and elucidate the underlying genetic mechanisms. We further highlight the roles of non-homologous end joining and translesion synthesis pathways in Zeocin-induced mutations, demonstrating that such treatments can rapidly confer drug resistance to the cells. Overall, our research enhances the understanding of how yeast genomes evolve under various conditions and provides guidance for developing more effective mutagenesis and breeding techniques.

Many species of proteobacterial methane-consuming bacteria (methanotrophs) form a hauberk-like envelope represented by a surface (S-) layer protein (SLP) matrix. While several proteins were predicted to be associated with the cell surface, the composition and function of the hauberk matrix remained elusive. Here, we report the identification of the genes encoding the hauberk-forming proteins in two gamma-proteobacterial (Type I) methanotrophs, Methylotuvimicrobium buryatense 5GB1 (EQU24_15540) and Methylotuvimicrobium alcaliphilum 20Z^R (MEALZ_0971 and MEALZ_0972). The proteins share 40% amino acid (AA) sequence identity with each other and are distantly related to the RsaA proteins from Caulobacter crescentus (20% AA sequence identity). Deletion of these genes resulted in loss of the characteristic hauberk pattern on the cell surface. A set of transcriptional fusions between the MEALZ_0971 and a superfolder green fluorescent protein (sfGFP) further confirmed its surface localization. The functional roles of the hauberk and cell-surface-associated proteins, including MEALZ_0971, MEALZ_0972, EQU24_15540, and a copper-induced CorA protein, were further investigated via gene expression studies and phenotypic tests. The hauberk core protein of M. alcaliphilum 20Z^R showed constitutive expression across 18 growth conditions with reduced growth at high salinity, high methanol, and metal-limited conditions, suggesting a role in cell-envelope stability and metal scavenging. Overall, understanding the genetics, composition, and cellular functions of S-layers contributes to our knowledge of methanotroph adaptation to environmental perturbations and opens a promising prospect for (nano)biotechnology applications.

Importance: Understanding the genetics, composition, and cellular functions of the cell envelope proteins, such as S-layers, contributes to our knowledge of microbial cell biology and stress responses and molecular adaptations to environmental perturbations. In addition, this study opens a promising prospect for (nano)biotechnology applications of methane-derived self-assembling protein materials.