Electrobiocorrosion by microbes without outer-surface cytochromes

Anaerobic microbial corrosion of iron-containing metals causes extensive economic damage.Some microbes are capable of direct metal-to-microbe electron transfer(electrobiocorrosion),but the prevalence of electrobiocorrosion among diverse methanogens and acetogens is poorly understood because of a lack of tools for their genetic manipulation.Previous studies have suggested that respiration with 316L stainless steel as the electron donor is indicative of electrobiocorrosion,because,unlike pure Fe^(0),316L stainless steel does not abiotically generate H2 as an intermediary electron carrier.Here,we report that all of the methanogens(Methanosarcina vacuolata,Methanothrix soehngenii,and Methanobacterium strain IM1)and acetogens(Sporomusa ovata and Clostridium ljungdahli)evaluated respired with pure Fe^(0)as the electron donor,but only M.vacuolata,Mx.soehngeni,and S.ovata were capable of stainless steel electrobiocorrosion.The electrobiocorrosive methanogens re-quired acetate as an additional energy source in order to produce methane from stainless steel.Cocultures of S.ovata and Mx.soehngeni demonstrated how acetogens can provide acetate to methanogens during corrosion.Not only was Meth-anobacterium strain IM1 not capable of electrobiocorrosion,but it also did not accept electrons from Geobacter metal-lireducens,an effective electron-donating partner for direct interspecies electron transfer to all methanogens that can directly accept electrons from Fe^(0).The finding that M.vacuolata,Mx.soehngeni,and S.ovata are capable of electrobiocorrosion,despite a lack of the outer-surface c-type cytochromes previously found to be important in other electrobiocorrosive microbes,demonstrates that there are multiple microbial strategies for making electrical contact with Fe^(0).

Rational design of unrestricted pRN1 derivatives and their application in the construction of a dual plasmid vector system for Saccharolobus islandicus

Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools,which include efficient genome editing,gene silencing,and robust protein expression systems.However,plasmid vectors constructed for this crenarchaeon thus far are based solely on the pRN2 cryptic plasmid.Although this plasmid coexists with pRN1 in its original host,early attempts to test pRN1-based vectors consistently failed to yield any stable host-vector system for Sa.islandicus.We hypothesized that this failure could be due to the occurrence of CRISPR immunity against pRN1 in this archaeon.We identified a putative target sequence in orf904 encoding a putative replicase on pRN1(target N1).Mutated targets(N1a,N1b,and N1c)were then designed and tested for their capability to escape the host CRISPR immunity by using a plasmid inter-ference assay.The results revealed that the original target triggered CRISPR immunity in this archaeon,whereas all three mutated targets did not,indicating that all the designed target mutations evaded host immunity.These mutated targets were then incorporated into orf904 individually,yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed(pN1aSD,pN1bSD,and pN1cSD).Sa.islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors,but pN1cSD lost the capability for replication.These results indicate that the missense mutations in the conserved helicase domain in pN1c inactivated the replicase.We further showed that pRN1-based and pRN2-based vectors were stably maintained in the archaeal cells either alone or in combination,and this yielded a dual plasmid system for genetic study with this important archaeal model.

O-glycosylation in viruses:A sweet tango

O-glycosylation is an ancient yet underappreciated protein posttranslational modification,on which many bacteria and viruses heavily rely to perform critical biological functions involved in numerous infectious diseases or even cancer.But due to the innate complexity of O-glycosylation,research techniques have been limited to study its exact role in viral attachment and entry,assembly and exit,spreading in the host cells,and the innate and adaptive immunity of the host.Recently,the advent of many newly developed methodologies(e.g.,mass spectrometry,chemical biology tools,and molecular dynamics simulations)has renewed and rekindled the interest in viral-related O-glycosylation in both viral proteins and host cells,which is further fueled by the COVID-19 pandemic.In this review,we summarize recent advances in viral-related O-glycosylation,with a particular emphasis on the mucin-type O-linkedα-N-acetylgalactosamine(O-GalNAc)on viral proteins and the intracellular O-linkedβ-N-acetylglucosamine(O-GlcNAc)modifications on host proteins.We hope to provide valuable insights into the development of antiviral reagents or vaccines for better prevention or treatment of infectious diseases.

Cultivating the uncultured:Harnessing the“sandwich agar plate”approach to isolate heme-dependent bacteria from marine sediment

In the classical microbial isolation technique,the isolation process inevitably destroys all microbial interactions and thus makes it difficult to culture the many microorganisms that rely on these interactions for survival.In this study,we designed a simple coculture technique named the“sandwich agar plate method,”which maintains microbial interactions throughout the isolation and pure culture processes.The total yield of uncultured species in sandwich agar plates based on eight helper strains was almost 10-fold that of the control group.Many uncultured species displayed commensal lifestyles.Further study found that heme was the growth-promoting factor of some marine commensal bacteria.Subsequent genomic analysis revealed that heme auxotrophies were common in various biotopes and prevalent in many uncultured microbial taxa.Moreover,our study supported that the survival strategies of heme auxotrophy in different habitats varied considerably.These findings highlight that cocultivation based on the“sandwich agar plate method”could be developed and used to isolate more uncultured bacteria.

RsaL is a self-regulatory switch that controls alternative biosynthesis of two AHL-type quorum sensing signals in Pseudomonas aeruginosa PA1201

Pseudomonas aeruginosa is a ubiquitous and metabolically versatile microorganism naturally found in soil and water.It is also an opportunistic pathogen in plants,insects,animals,and humans.In response to increasing cell density,P.aeruginosa uses two acylhomoserine lactone(AHL)quorum-sensing(QS)signals(i.e.,N-3-oxo-dodecanoyl homoserine lactone[3-oxo-C12-HSL]and Nbutanoyl-homoserine lactone[C4-HsL]),which regulate the expression of hundreds of genes.However,how the biosynthesis of these two QS signals is coordinated remains unknown.We studied the regulation of these two QS signals in the rhizosphere strain PA1201.PA1201 sequentially produced 3-oxo-C12-HSL and C4-HSL at the early and late growth stages,respectively.The highest 3-oxo-C12-HSL-dependent elastase activity was observed at the early stage,while the highest C4-HSL-dependent rhamnolipid production was observed at the late stage.The atypical regulator RsaL played a pivotal role in coordinating 3-oxo-C12-HSL and C4-HSL biosynthesis and QS-associated virulence.RsaL repressed las/transcription by binding the-10 and-35 boxes of the lasl promoter.In contrast,RsaL activated rhll transcription by binding the region encoding the 5'-untranslated region of the rhll mRNA.Further,RsaL repressed its own expression by binding a nucleotide motif located in the-35 box of the rsaL promoter.Thus,RsaL acts as a molecular switch that coordinates the sequential biosynthesis of AHL QS signals and differential virulence in PA1201.Finally,C4-HSL activation by RsaL was independent of the Las and Pseudomonas quinolone signal(PQS)QS signaling systems.Therefore,we propose a new model of the QS regulatory network in PA1201,in which RsaL represents a superior player acting at the top of the hierarchy.

Fight or flee,a vital choice for Clostridioides difficile

Clostridioides difficile is a leading cause of healthcare-associated infections,causing billions of economic losses every year.Its symptoms range from mild diarrhea to life-threatening damage to the colon.Transmission and recurrence of c.difficile infection(CDl)are mediated by the metabolically dormant spores,while the virulence of C.difficile is mainly due to the two large clostridial toxins,TcdA and TcdB.Producing toxins or forming spores are two different strategies for C.difficile to cope with harsh environmental conditions.It is of great significance to understand the molecular mechanisms for C.difficile to skew to either of the cellular processes.Here,we summarize the current understanding of the regulation and connections between toxin production and sporulation in C.difficile and further discuss the potential solutions for yet-to-be-answered questions.

Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins inKluyveromyces marxianus

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins.Improving the yield in K.marxianus remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering.To address these issues,linear and circular yeast artificial chromosomes of K.marxianus(KmYACs)were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K.marxianus.These modules contained up to seven genes with a maximum size of 15 kb.KmYACs carried telomeres either from K.marxianus or Tetrahymena.KmYACs were transferred successfully into K.marxianus and stably propagated without affecting the normal growth of the host,regardless of the type of telomeres and configurations of KmYACs.KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins.In high-density fermentation,the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l,the highest reported level to date in K.marxianus.Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis,enhanced flux entering the tricarboxylic acid cycle,and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins.Consistently,supplementing lysine or arginine further improved the yield.Therefore,KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research.Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins,and this strategy may be applied to optimize other microbial cell factories.

Insertion sequence transposition activates antimycobacteriophage immunity through an Isr2-silenced lipid metabolism gene island

Insertion sequences(ISs)exist widely in bacterial genomes,but their roles in the evolution of bacterial antiphage defense remain to be clarified.Here,we report that,under the pressure of phage infection,the IS1o96 transposition of Mycobacterium smegmatis into the Isr2 gene can occur at high frequencies,which endows the mutant mycobacterium with a broad-spectrum antiphage ability.Lsr2 functions as a negative regulator and directly silences expression of a gene island composed of 11 lipid metabolism-related genes.The complete or partial loss of the gene island leads to a significant decrease of bacteriophage adsorption to the mycobacterium,thus defending against phage infection.Strikingly,a phage that has evolved mutations in two tail-filament genes can re-escape from the Isr2 inactivation-triggered host defense.This study uncovered a new signaling pathway for activating antimycobacteriophage immunity by Is transposition and provided insight into the natural evolution of bacterial antiphage defense.

Ranking environmental and edaphic attributes driving soil microbial community structure and activity with special attention to spatial and temporal scales

The incredibly complex soil microbial communities at small scales make their analysis and identification of reasons for the observed structures challenging.Microbial community structure is mainly a result of the inoculum(dispersal),the selective advantages of those organisms under the habitat-based environmental attributes,and the ability of those colonizers to sustain themselves over time.Since soil is protective,and its microbial inhabitants have long adapted to varied soil conditions,significant portions of the soil microbial community structure are likely stable.Hence,a substantial portion of the community will not correlate to often measured soil attributes.We suggest that the drivers be ranked on the basis of their importance to the fundamental needs of the microbes:(i)those that supply energy,i.e.,organic carbon and electron acceptors;(i)environmental effectors or stressors,i.e.,pH,salt,drought,and toxic chemicals;(ii)macro-organism associations,i.e.,plants and their seasonality,animals and their fecal matter,and soil fauna;and(iv)nutrients,in order,N,P,and probably of lesser importance,other micronutrients,and metals.The relevance of drivers also varies with spatial and time scales,for example,aggregate to field to regional,and persistent to dynamic populations to transcripts,and with the extent of phylogenetic difference,hence phenotypic differences in organismal groups.We present a summary matrix to provide guidance on which drivers are important for particular studies,with special emphasis on a wide range of spatial and temporal scales,and illustrate this with genomic and population(rRNA gene)data from selected studies.

Understanding the rapid spread of antimicrobial resistance genes mediated by Is26

Insertion sequences(ISs)promote the transmission of antimicrobial resistance genes(ARGs)across bacterial populations.However,their contributions and dynamics during the transmission of resistance remain unclear.In this study,we selected is26 as a representative transposable element to decipher the relationship between ISs and ARGs and to investigate their transfer features and transmission trends.We retrieved 2656 translocatable IS26-bounded units with ARGs(tiS26-bUs-ARGs)in complete bacterial genomes from the NCBI RefSeq database.In total,124 ARGs spanning 12 classes of antibiotics were detected,and the average contribution rate of IS26 to these genes was 41.2%.We found that IS26-bounded units(IS26-bUs)mediated extensive ARG dissemination within the bacteria of the Gammaproteobacteria class,showing strong transfer po-tential between strains,species,and even phyla.The Is26-bUs expanded in bacterial populations over time,and their temporal expansion trend was significantly correlated with antibiotic usage.This wide dissemination could be due to the nonspecific target site preference of is26.Finally,we experimentally confirmed that the introduction of a single copy of is26 could lead to the formation of a composite transposon mediating the transmission of“passenger”genes.These observations extend our knowledge of the Is26 and provide new insights into the mediating role of ISs in the dissemination of antibiotic resistance.

The global regulation of c-di-GMP and cAMP in bacteria

Nucleotide second messengers are highly versatile signaling molecules that regulate a variety of key biological processes in bacteria.The best-studied examples are cyclic AMP(cAMP)and bis-(3'-5')-cyclic dimeric guanosine monophosphate(c-di-GMP),which both act as global regulators.Global regulatory frameworks of c-di-GMP and cAMP in bacteria show several parallels but also significant variances.In this review,we llustrate the global regulatory models of the two nucleotide second messengers,compare the different regulatory frameworks between c-di-GMP and cAMP,and discuss the mechanisms and physiological significance of cross-regulation between c-di-GMP and cAMP.c-di-GMP responds to numerous signals de-pendent on a great number of metabolic enzymes,and it regulates various signal transduction pathways through its huge number of effectors with varying activities.In contrast,due to the limited quantity,the cAMP metabolic enzymes and its major effector are regulated at different levels by diverse signals.cAMP performs its global regulatory function primarily by controlling the transcription of a large number of genes via cAMP receptor protein(CRP)in most bacteria.This review can help us understand how bacteria use the two typical nucleotide second messengers to effectively coordinate and integrate various physiological processes,providing theoretical guidelines for future research.

Whyand how tousethe SeqCode

The SeqCode,formally called the Code of Nomenclature of Prokaryotes Described from Sequence Data,is a new code of nomenclature in which genome sequences are the nomenclatural types for the names of prokaryotic species.While similar to the International Code of Nomenclature of Prokaryotes(ICNP)in structure and rules of priority,it does not require the deposition of type strains in international culture collections.Thus,it allows for the formation of permanent names for uncultured prokaryotes whose nearly complete genome sequences have been obtained directly from environmental DNA as well as other prokaryotes that cannot be deposited in culture collections.Because the diversity of uncultured prokaryotes greatly exceeds that of readily culturable prokaryotes,the SeqCode is the only code suitable for naming the majority of prokaryotic species.The start date of the SeqCode was January 1,2022,and the online Registry(https://seqco.del)was created to ensure valid publication of names.The SeqCode recognizes all names validly published under the ICNP before 2022.After that date,names validly published under the SeqCode compete with ICNP names for priority.As a result,species can have only one name,either from the SeqCode or ICNP,enabling effective communication and the creation of unified taxonomies of uncultured and cultured prokaryotes.The SeqCode is administered by the SeqCode Committee,which is comprised of the SeqCode Community and elected administrative components.Anyone with an interest in the systematics of prokaryotes is encouraged to join the SeqCode Community and participate in the development of this resource.

Reviewer Acknowledgments

mLife thanks the following reviewers for their great support and contribution in 2023.

Genomic investigation and nationwide tracking of pediatric invasive nontyphoidal Salmonella in China

Impact Statement。Invasive nontyphoidal Salmonella(iNTS)causes significant concern with~15%morbidity,affecting populations mainly in African countries.

Variation in resource competition traits among Microcystis strains is affected by their microbiomes

Freshwater harmful algal blooms are often dominated by Microcystis,a phylogenetically cohesive group of cyanobacteria marked by extensive genetic and physiological diversity.We have previously shown that this genetic diversity and the presence of a microbiome of heterotrophic bacteria influences competitive interactions with eukaryotic phytoplankton.In this study,we sought to explain these observations by characterizing Monod equation parameters for resource usage(maximum growth rateμmax,half-saturation value for growth Ks,and quota)as a function of N and P levels for four strains(NIES-843,PCC 9701,PCC 7806[WT],and PCC 7806ΔmcyB)in presence and absence of a microbiome derived from Microcystis isolated from Lake Erie.Results indicated limited differences in maximum growth rates but more pronounced differences in half-saturation values among Microcystis strains.The largest impact of the microbiome was reducing the minimal nitrogen concentration sustaining growth and reducing half saturation values,with variable results depending on the Microcystis strain.Microcystis strains also differed from each other in their N and P quotas and the extent to which microbiome presence affected them.Our data highlight the importance of the microbiome in altering Microcystis-intrinsic traits,strain competitive hierarchies,and thus bloom dynamics.As quota,μmax,and Ks are commonly used in models for harmful algal blooms,our data suggest that model improvement may be possible by incorporating genotype dependencies of resource-use parameters.

The diversity and ecological significance of microbial traits potentially involved in B_(12) biosynthesis in the global ocean

Cobalamin(B_(12)),an essential nutrient and growth cofactor for many living organisms on Earth,can be fully synthesized only by selected prokaryotes in nature.Therefore,microbial communities related to B_(12) biosynthesis could serve as an example subsystem to disentangle the underlying ecological mechanisms balancing the function and taxonomic make-up of complex functional assemblages.By anchoring microbial traits potentially involved in B_(12) biosynthesis,we depict the biogeographic patterns of B_(12) biosynthesis genes and the taxa harboring them in the global ocean,despite the limitations of detecting de novo B_(12) synthesizers via metagenomes alone.Both the taxonomic and functional composition of B_(12) biosynthesis genes were strongly shaped by depth,differentiating the epipelagic zones from the mesopelagic layers.Functional genes related to B_(12) biosynthesis were relatively stably distributed across different oceans,but the taxa harboring them varied considerably,showing clear functional redundancy among microbial systems.Microbial taxa carrying B_(12) biosynthesis genes in the surface water were influenced by environmental factors such as temperature,oxygen,and nitrate.However,the composition of functional genes was only weakly associated with these environmental factors.Null model analyses demonstrated that determinism governed the variations in B_(12) biosynthesis genes,whereas a higher degree of stochasticity was associated with taxonomic variations.Significant associations were observed between the chlorophyll a concentration and B_(12) biosynthesis,confirming its importance in primary production in the global ocean.The results of this study reveal an essential ecological mechanism governing the assembly of microbes in nature:the environment selects for function rather than taxonomy;functional redundancy underlies stochastic community assembly.

Gut microbiota research nexus:One Health relationshipbetween human,animal,and environmental resistomes

The emergence and rapid spread of antimicrobial resistance is of global public health concern.The gut microbiota harboring diverse commensal and opportunistic bacteria that can acquire resistance via horizontal and vertical gene transfers is considered an important reservoir and sink of antibiotic resistance genes(ARGs).In this review,we describe the reservoirs of gut ARGs and their dynamics in both animals and humans,use the One Health perspective to track the transmission of ARG-containing bacteria between humans,animals,and the environment,and assess the impact of antimicrobial resistance on human health and socioeconomic development.The gut resistome can evolve in an environment subject to various selective pressures,including antibiotic administration and environmental and lifestyle factors(e.g.,diet,age,gender,and living conditions),and interventions through probiotics.Strategies to reduce the abundance of clinically relevant antibiotic-resistant bacteria and their resistance determinants in various environmental niches are needed to ensure the mitigation of acquired antibiotic resistance.With the help of effective measures taken at the national,local,personal,and intestinal management,it will also result in preventing or minimizing the spread of infectious diseases.This review aims to improve our understanding of the correlations between intestinal microbiota and antimicrobial resistance and provide a basis for the development of management strategies to mitigate the antimicrobial resistance crisis.

Resilience and functional redundancy of methanogenic digestion microbiome safeguard recovery of methanogenesis activity under the stress induced by microplastics

Microplastics and nanoplastics are emerging pollutants that substantially influence biological element cycling in natural ecosystems.Plastics are also prevalent in sewage,and they accumulate in waste-activated sludge(WAS).However,the impacts of plastics on the methanogenic digestion of WAS and the underpinning microbiome remain underexplored,particularly during long-term operation.In this study,we found that short-term exposure to individual microplastics and nanoplastics(polyethylene,polyvinyl chloride,polystyrene,and polylactic acid)at a low concentration(10 particles/g sludge)slightly enhanced methanogenesis by 2.1%−9.0%,whereas higher levels(30−200 particles/g sludge)suppressed methanogenesis by 15.2%−30.1%.Notably,the coexistence of multiple plastics,particularly at low concentrations,showed synergistic suppression of methanogenesis.Unexpectedly,methanogenesis activity completely recovered after long-term exposure to plastics,despite obvious suppression of methanogenesis by initial plastic exposure.The inhibition of methanogenesis by plastics could be attributed to the stimulated generation of reactive oxygen species.The stress induced by plastics dramatically decreased the relative abundance of methanogens but showed marginal influence on putative hydrolytic and fermentation populations.Nonetheless,the digestion sludge microbiome exhibited resilience and functional redundancy,contributing to the recovery of methanogenesis during the long-term operation of digesters.Plastics also increased the complexity,modularity,and negative interaction ratios of digestion sludge microbiome networks,but their influence on community assembly varied.Interestingly,a unique plastisphere was observed,the networks and assembly of which were distinct from the sludge microbiome.Collectively,the comprehensive evaluation of the influence of microplastics and nanoplastics on methanogenic digestion,together with the novel ecological insights,contribute to better understanding and manipulating this engineered ecosystem in the face of increasing plastic pollution.

The bZIP transcription factor ATF1 regulates blue light and oxidative stress responses in Trichoderma guizhouense

In several filamentous fungi,incident light and environmental stress signaling share the mitogen-activated protein kinase(MAPK)HOG(SAK)pathway.It has been revealed that short-term illumination with blue light triggers the activation of the HOG pathway in Trichoderma spp.In this study,we demonstrate the crucial role of the basic leucine zipper transcription factor ATF1 in blue light responses and signaling downstream of the MAPK HOG1 in Trichoderma guizhouense.The lack of ATF1 severely impaired photoconidiation and delayed vegetative growth and conidial germination.Upon blue light or H2O2 stimuli,HOG1 interacted with ATF1 in the nucleus.Genome-wide transcriptome analyses revealed that 61.8%(509 out of 824)and 85.2%(702 out of 824)of blue light-regulated genes depended on ATF1 and HOG1,respectively,of which 58.4%(481 out of 824)were regulated by both of them.Our results also show that blue light promoted conidial germination and HOG1 and ATF1 played opposite roles in controlling conidial germination in the dark.Additionally,the lack of ATF1 led to reduced oxidative stress resistance,probably because of the downregulation of catalase-encoding genes.Overall,our results demonstrate that ATF1 is the downstream component of HOG1 and is responsible for blue light responses,conidial germination,vegetative growth,and oxidative stress resistance in T.guizhouense.

Sequence-discrete species for prokaryotes and other microbes:A historical perspective and pending issues

Whether prokaryotes,and other microorganisms,form distinct clusters that can be recognized as species remains an issue of paramount theoretical as well as practical consequence in identifying,regulating,and communicating about these organisms.In the past decade,comparisons of thousands of genomes of isolates and hundreds of metagenomes have shown that prokaryotic diversity may be predominantly organized in such sequence-discrete clusters,albeit organisms of intermediate relatedness between the identified clusters are also frequently found.Accumulating evidence suggests,however,that the latter“intermediate”organisms show enough ecological and/or functional distinctiveness to be considered different species.Notably,the area of discontinuity between clusters often—but not always—appears to be around 85%–95%genome-average nucleotide identity,consistently among different taxa.More recent studies have revealed remarkably similar diversity patterns for viruses and microbial eukaryotes as well.This high consistency across taxa implies a specific mechanistic process that underlies the maintenance of the clusters.The underlying mechanism may be a substantial reduction in the efficiency of homologous recombination,which mediates(successful)horizontal gene transfer,around 95%nucleotide identity.Deviations from the 95%threshold(e.g.,species showing lower intraspecies diversity)may be caused by ecological differentiation that imposes barriers to otherwise frequent gene transfer.While this hypothesis that clusters are driven by ecological differentiation coupled to recombination frequency(i.e.,higher recombination within vs.between groups)is appealing,the supporting evidence remains anecdotal.The data needed to rigorously test the hypothesis toward advancing the species concept are also outlined.