Biochemical mechanism of phosphorus limitation impairing nitrogen fixation in diazotrophic bacterium <i>Klebsiella variicola</i> W12

Introduction: Biological nitrogen (N) fixation (BNF) plays a key role in nitrogen supply in agricultural and natural ecosystems. Harnessing BNF can substantially reduce dependence on chemical fertilizer in agroecosystems and hence can contribute to sustainable agriculture. However, a number of field studies have demonstrated that BNF can be largely suppressed in phosphorus (P)-deficient environments, while the underlying mechanism is not well understood.Materials &amp; Methods: In this study, comparative proteomics and lipidomics analyses were conducted on a diazotrophic bacterium Klebsiella variicola W12 under P-deficient and P-replete conditions to gain insight into how P availability affects N fixation.Results: Under P deficiency, N fixation activity of K. variicola W12 was severely repressed. In response to P limitation, the bacterium synthesized P-free ornithine lipids to replace glycerophospholipids in its membrane to reduce cellular demand for P. Comparative proteomics showed that P limitation resulted in upregulation of the PhoBR two-component system, a range of organic and inorganic P uptake and transport systems, while nitrogenase and N-fixation-related transcriptional regulators NifL and NifA were downregulated.Conclusion: These results revealed lipid renovation as an adaptation strategy for N2-fixing microbes to survive under P stress and provided biochemical evidence on how P availability regulates BNF. A conceptual model of N–P coupling at the microbial metabolism level is therefore proposed. Our study provides a simple yet plausible explanation of how P deficiency suppresses BNF observed in the field and highlights the importance of regulating P availability to maximize the potential of BNF in agroecosystems for agriculture sustainable production

Biochemical mechanism of phosphorus limitation impairing nitrogen fixation in diazotrophic bacterium <i>Klebsiella variicola</i> W12

Introduction: Biological nitrogen (N) fixation (BNF) plays a key role in nitrogen supply in agricultural and natural ecosystems. Harnessing BNF can substantially reduce dependence on chemical fertilizer in agroecosystems and hence can contribute to sustainable agriculture. However, a number of field studies have demonstrated that BNF can be largely suppressed in phosphorus (P)-deficient environments, while the underlying mechanism is not well understood.Materials &amp; Methods: In this study, comparative proteomics and lipidomics analyses were conducted on a diazotrophic bacterium Klebsiella variicola W12 under P-deficient and P-replete conditions to gain insight into how P availability affects N fixation.Results: Under P deficiency, N fixation activity of K. variicola W12 was severely repressed. In response to P limitation, the bacterium synthesized P-free ornithine lipids to replace glycerophospholipids in its membrane to reduce cellular demand for P. Comparative proteomics showed that P limitation resulted in upregulation of the PhoBR two-component system, a range of organic and inorganic P uptake and transport systems, while nitrogenase and N-fixation-related transcriptional regulators NifL and NifA were downregulated.Conclusion: These results revealed lipid renovation as an adaptation strategy for N2-fixing microbes to survive under P stress and provided biochemical evidence on how P availability regulates BNF. A conceptual model of N–P coupling at the microbial metabolism level is therefore proposed. Our study provides a simple yet plausible explanation of how P deficiency suppresses BNF observed in the field and highlights the importance of regulating P availability to maximize the potential of BNF in agroecosystems for agriculture sustainable production

Cyanophage MazG is a pyrophosphohydrolase but unable to hydrolyse magic spot nucleotides

Bacteriophage possess a variety of auxiliary metabolic genes (AMGs) of bacterial origin. These proteins enable them to maximise infection efficiency, subverting bacterial metabolic processes for the purpose of viral genome replication and synthesis of the next generation of virion progeny. Here, we examined the enzymatic activity of a cyanophage MazG protein – a putative pyrophosphohydrolase previously implicated in regulation of the stringent response via reducing levels of the central alarmone molecule (p)ppGpp. We demonstrate however, that the purified viral MazG shows no binding or hydrolysis activity against (p)ppGpp. Instead, dGTP and dCTP appear to be the preferred substrates of this protein, consistent with a role preferentially hydrolysing deoxyribonucleotides from the high GC content host Synechococcus genome. This showcases a new example of the fine‐tuned nature of viral metabolic processes

Comparative genomics of bacteriophage of the genus Seuratvirus

Despite being more abundant and having smaller genomes than their bacterial host, relatively few bacteriophages have had their genomes sequenced. Here, we isolated 14 bacteriophages from cattle slurry and performed de novo genome sequencing, assembly, and annotation. The commonly used marker genes polB and terL showed these bacteriophages to be closely related to members of the genus Seuratvirus. We performed a core-gene analysis using the 14 new and four closely related genomes. A total of 58 core genes were identified, the majority of which has no known function. These genes were used to construct a core-gene phylogeny, the results of which confirmed the new isolates to be part of the genus Seuratvirus and expanded the number of species within this genus to four. All bacteriophages within the genus contained the genes queCDE encoding enzymes involved in queuosine biosynthesis. We suggest these genes are carried as a mechanism to modify DNA in order to protect these bacteriophages against host endonucleases

Coordinated transcriptional response to environmental stress by a Synechococcus virus

Viruses are a major control on populations of microbes. Often, their virulence is examined in controlled laboratory conditions. Yet, in nature, environmental conditions lead to changes in host physiology and fitness that may impart both costs and benefits on viral success. Phosphorus (P) is a major abiotic control on the marine cyanobacterium Synechococcus. Some viruses infecting Synechococcus have acquired, from their host, a gene encoding a P substrate binding protein (PstS), thought to improve virus replication under phosphate starvation. Yet, pstS is uncommon amongst cyanobacterial viruses. Thus, we asked how infections with viruses lacking PstS are affected by P scarcity. We show that production of infectious virus particles of such viruses is reduced in low P conditions. However, this reduction in progeny is not caused by impaired phage genome replication, thought to be a major sink for cellular phosphate. Instead, transcriptomic analysis showed that under low P conditions a PstS-lacking cyanophage increased the expression of a specific gene set that included mazG, hli2, and gp43 encoding a pyrophosphatase, a high-light inducible proteinand DNA polymerase respectively. Moreover, several of the upregulated genes were controlled by the hosts phoBR two-component system. We hypothesise that recycling and polymerization of nucleotides liberates free phosphate and thus allows viral morphogenesis, albeit at lower rates than when phosphate is replete or when phages encode pstS. Together, our data shows how phage genomes, lacking obvious P-stress related genes, have evolved to exploit their host’s environmental sensing mechanisms to coordinate their own gene expression in response to resource limitation

A new family of globally distributed lytic roseophages with unusual deoxythymidine to deoxyuridine substitution

Marine bacterial viruses (bacteriophages) are abundant biological entities that are vital for shaping microbial diversity, impacting marine ecosystem function, and driving host evolution.1, 2, 3 The marine roseobacter clade (MRC) is a ubiquitous group of heterotrophic bacteria[4],[5] that are important in the elemental cycling of various nitrogen, sulfur, carbon, and phosphorus compounds.6, 7, 8, 9, 10 Bacteriophages infecting MRC (roseophages) have thus attracted much attention and more than 30 roseophages have been isolated,11, 12, 13 the majority of which belong to the N4-like group (Podoviridae family) or the Chi-like group (Siphoviridae family), although ssDNA-containing roseophages are also known.[14] In our attempts to isolate lytic roseophages, we obtained two new phages (DSS3_VP1 and DSS3_PM1) infecting the model MRC strain Ruegeria pomeroyi DSS-3. Here, we show that not only do these phages have unusual substitution of deoxythymidine with deoxyuridine (dU) in their DNA, but they are also phylogenetically distinct from any currently known double-stranded DNA bacteriophages, supporting the establishment of a novel family (“Naomiviridae”). These dU-containing phages possess DNA that is resistant to the commonly used library preparation method for metagenome sequencing, which may have caused significant underestimation of their presence in the environment. Nevertheless, our analysis of Tara Ocean metagenome datasets suggests that these unusual bacteriophages are of global importance and more diverse than other well-known bacteriophages, e.g., the Podoviridae in the oceans, pointing to an overlooked role for these novel phages in the environment

The long and short of it : benchmarking viromics using Illumina, Nanopore and PacBio sequencing technologies

Viral metagenomics has fuelled a rapid change in our understanding of global viral diversity and ecology. Long-read sequencing and hybrid assembly approaches that combine long- and short-read technologies are now being widely implemented in bacterial genomics and metagenomics. However, the use of long-read sequencing to investigate viral communities is still in its infancy. While Nanopore and PacBio technologies have been applied to viral metagenomics, it is not known to what extent different technologies will impact the reconstruction of the viral community. Thus, we constructed a mock bacteriophage community of previously sequenced phage genomes and sequenced them using Illumina, Nanopore and PacBio sequencing technologies and tested a number of different assembly approaches. When using a single sequencing technology, Illumina assemblies were the best at recovering phage genomes. Nanopore- and PacBio-only assemblies performed poorly in comparison to Illumina in both genome recovery and error rates, which both varied with the assembler used. The best Nanopore assembly had errors that manifested as SNPs and INDELs at frequencies 41 and 157 % higher than found in Illumina only assemblies, respectively. While the best PacBio assemblies had SNPs at frequencies 12 and 78 % higher than found in Illumina-only assemblies, respectively. Despite high-read coverage, long-read-only assemblies recovered a maximum of one complete genome from any assembly, unless reads were down-sampled prior to assembly. Overall the best approach was assembly by a combination of Illumina and Nanopore reads, which reduced error rates to levels comparable with short-read-only assemblies. When using a single technology, Illumina only was the best approach. The differences in genome recovery and error rates between technology and assembler had downstream impacts on gene prediction, viral prediction, and subsequent estimates of diversity within a sample. These findings will provide a starting point for others in the choice of reads and assembly algorithms for the analysis of viromes

A novel class of sulfur-containing aminolipids widespread in marine roseobacters

Marine roseobacter group bacteria are numerically abundant and ecologically important players in ocean ecosystems. These bacteria are capable of modifying their membrane lipid composition in response to environmental change. Remarkably, a variety of lipids are produced in these bacteria, including phosphorus-containing glycerophospholipids and several amino acid-containing aminolipids such as ornithine lipids and glutamine lipids. Here, we present the identification and characterization of a novel sulfur-containing aminolipid (SAL) in roseobacters. Using high resolution accurate mass spectrometry, a SAL was found in the lipid extract of Ruegeria pomeroyi DSS-3 and Phaeobacter inhibens DSM 17395. Using comparative genomics, transposon mutagenesis and targeted gene knockout, we identified a gene encoding a putative lyso-lipid acyltransferase, designated salA, which is essential for the biosynthesis of this SAL. Multiple sequence analysis and structural modeling suggest that SalA is a novel member of the lysophosphatidic acid acyltransferase (LPAAT) family, the prototype of which is the PlsC acyltransferase responsible for the biosynthesis of the phospholipid phosphatidic acid. SAL appears to play a key role in biofilm formation in roseobacters. salA is widely distributed in Tara Oceans metagenomes and actively expressed in Tara Oceans metatranscriptomes. Our results raise the importance of sulfur-containing membrane aminolipids in marine bacteria

A novel ATP dependent dimethylsulfoniopropionate lyase in bacteria that releases dimethyl sulfide and acryloyl-CoA

Dimethylsulfoniopropionate (DMSP) is an abundant and ubiquitous organosulfur molecule in marine environments with important roles in global sulfur and nutrient cycling. Diverse DMSP lyases in some algae, bacteria and fungi cleave DMSP to yield gaseous dimethyl sulfide (DMS), an infochemical with important roles in atmospheric chemistry. Here we identified a novel ATP-dependent DMSP lyase, DddX. DddX belongs to the acyl-CoA synthetase superfamily and is distinct from the eight other known DMSP lyases. DddX catalyses the conversion of DMSP to DMS via a two-step reaction: the ligation of DMSP with CoA to form the intermediate DMSP-CoA, which is then cleaved to DMS and acryloyl-CoA. The novel catalytic mechanism was elucidated by structural and biochemical analyses. DddX is found in several Alphaproteobacteria, Gammaproteobacteria and Firmicutes, suggesting that this new DMSP lyase may play an overlooked role in DMSP/DMS cycles

ISMEJ-D-23-00494

&lt;p&gt;Data files accompanying the publication under ISMEJ-D-23-00494 number&lt;/p&gt