microLife

Archaea and bacteria routinely live side by side in microbial communities and must interact at least on occasion. Whether such cross-Domain interactions are dominated by mutual disregard, co-operation, or conflict remains fundamentally unknown. One potential window into archaeal-bacterial conflict is to ask whether some of the molecular weapons bacteria wield to kill other bacteria are present in archaea, and vice versa. Here, to start to address this question, we carry out a phylogenomic survey of bacteriocins in archaeal genomes and archaeocins in bacterial genomes. We find that more than 20% of known bacteriocins - proteins deployed by bacteria against other bacteria - have at least one homolog in archaea. Typically, these archaeal homologs are related to bacteriocins targeting (and encoded by) monoderm bacteria. Based on conservation of functionally critical residues, protein structure, and accessory genes critical for bacteriocin biosynthesis, we highlight homologs of subtilosin A, encoded in some Thermococcus archaea, as promising candidates for experimental follow- up work. We also show that halocin C8, originally described in Natrinema archaea, is comparatively common in bacterial genomes, including a number of skin-resident Staphylococcus species. Our results suggest that bacteriocins/archaeocins are shared across Domain boundaries with some regularity. While many instances are phylogenetically isolated - raising doubts about their functional importance and integration into host physiology - some bacteriocins are present in multiple related genomes and embedded in broader biosynthetic gene clusters that are also found in the original producers, suggesting that archaea and bacteria periodically use the same weapon systems in conflicts with other microbes. Further study of these systems might elucidate cross-Domain conflict and the nature of archaeal-bacterial interactions in different environments.

The cell envelope of bacteria and fungi is a major source of microbial-associated molecular patterns (MAMPs), which are recognised by multiple host pattern recognition receptors (PRRs) such as TLRs and NODs. The key role of this recognition in protecting against infection and regulating diverse physiological processes has been studied for decades. In contrast, although the archaea are growingly recognised as an important component of the animal and human microbiome, their interaction with the innate immune system has been largely understudied. Here, we asked whether the archaeal cell envelope acts as MAMP. We selected 15 archaeal species from different environments, including the human microbiome, and harbouring diverse envelope characteristics, and we tested their ability to activate innate immune responses in human and murine immune cells. We first show that archaeal cells activate the innate immune transcription factor NF-kB and induce IL-6 secretion. Using whole archaeal cells, supernatants, and purified envelope components, we then find that none of the main PRR receptors which recognise the bacterial envelope (TLR4, TLR5, NOD1 and NOD2) are involved in this activation. We only detected activation of TLR2, regardless of the environmental origins of the archaea tested, although with overall lower activity compared to bacteria. We further demonstrate that different TLR2 heterodimers are involved, depending on the species, although the archaeal agonist remains unknown. Altogether, our results highlight a surprising paucity of interaction of archaeal cell envelope components with host PPRs, in contrast to bacteria, and open new avenues of research toward further investigation of archaea/host interplay.

ABSTRACT: The bacterial small RNA (sRNA) OxyS is expressed in Escherichia coli during oxidative stress. The sRNA OxyS enhances cell survival by controlling genes involved in the regulation of hydrogen peroxide (H2O2) and iron-sulfur (Fe-S) cluster formation. Here, we used the MS2 affinity purification coupled with RNA sequencing (MAPS) technique to identify new target mRNAs of the sRNA OxyS. Our analysis revealed a significant enrichment of mepS mRNA, which encodes a peptidoglycan endopeptidase that promotes cell growth. Our results confirm a previous report on the sRNA OxyS repressing the translation of mepS. We also found that an {Delta}oxyS background facilitates the emergence of mutations, conferring increased resistance to the last-resort antibiotics polymyxin B and E (colistin), but only in the presence of the target mepS gene. This suggests that the translation repression of mepS by OxyS could prevent mutations in bacterial DNA during H2O2-induced oxidative stress. Moreover, we show that adding the antioxidant thiourea or sequestering iron in the {Delta}oxyS background effectively reduces the emergence of resistance against both polymyxin B and colistin. These results suggest that reactive oxygen species (ROS), in conjunction with intracellular iron, play a key role in driving the emergence of antibiotic resistance. Overall, our work underlines a mechanism of antimicrobial emergence implicating oxidative stress, intracellular Fe, and cell wall remodeling in E. coli. IMPORTANCE: This study uncovers an underexplored link between peptidoglycan remodeling and oxidative stress responses during exposure to antibiotics. By elucidating how MepS and the sRNA OxyS interact in the presence of polymyxins and oxidative stress, our study suggests that MepS may exert an anti-mutator function. The repression of mepS translation by OxyS seems to limit the emergence of antibiotic resistance driven by DNA mutations. Together, these findings suggest cell wall remodeling and oxidative stress response pathways as promising targets to enhance antibiotic efficacy and limit the emergence of resistance.

Escherichia coli is a well-studied organism with extensive genomic and proteomic data. This study examines how gene loss reallocates cellular resources and impacts fitness. Genes were classified based on fitness measurements as essential, important, mean-effect, or fitness-enhancing. Using proteomic data, we analyzed the relationship between protein production cost and fitness, finding that genes with a high proteomic mass fraction are more likely to affect fitness, while fitness-enhancing deletions rarely improve fitness by reducing proteomic burden. We calculated the cumulative of proteome fractions encoded by genes classified as mean-effect and compared it with the results from the ME-model simulations. The mean-effect category constitutes 31-75% of the proteome, with the highest proportion LB, while enrichment analysis of core mean-effect genes highlighted transmembrane transport as the main functional category. Furthermore, we identified a subset of genes whose deletion increased fitness compared to the mean; they generally have low expression, and many have unknown functions. AI-assisted structural analyses identified domains and conserved features compatible with DNA-binding proteins, suggesting that some may represent putative transcriptional regulators requiring further validation. RpoS, stress sigma factor controlling up to 15% of the proteome is one of the transcriptional regulators in the fitness-enhancing category. Our findings suggest that the cost of being a generalist is linked to transcriptional regulation, while molecular transport represents a high burden for nutrient readiness. ImportanceThis study provides new insights into how gene loss benefits bacteria by identifying gene categories and their associated protein fractions whose disruption does not impose large fitness penalties. Additionally, it uncovers specific fitness-enhancing genes and generates hypotheses based on structural analyses for previously uncharacterized ones. Our findings suggest that several of these genes may encode putative transcriptional regulators, highlighting a potential role for regulatory complexity in cellular efficiency. By revealing how certain gene deletions enhance fitness and which gene categories are nonessential, this work advances our understanding of bacterial adaptation and genome streamlining. These insights have broad implications for evolutionary biology, metabolic engineering, and biotechnology, offering strategies to optimize microbial function by selectively reducing genetic and regulatory burden.

Bacterial cell wall (CW), primarily composed of the biopolymer peptidoglycan, serve as essential protective barriers against external stresses and the internal turgor pressure. The peptidoglycan (PG) biosynthetic pathway encompasses sequential enzymatic reactions in the cytoplasm and in the membrane that involve critical enzymes susceptible to antibiotic targeting. Virtually each step of the pathway is the target of a known antibiotic. Antibiotic-induced inhibition of PG assembly typically weakens the sacculus, often leading to cell lysis. However, the cascade of events that follow inhibition of a specific enzyme of the pathway, and how these culminate in cell death remain largely unknown. Here, we investigated the effects on growing Bacillus subtilis cells of two categories of CW antibiotics: inhibitors of the synthesis of soluble PG precursors in the cytoplasm (fosfomycin and D-cycloserine) and inhibitors of the polymerisation and crosslinking reactions at the outer leaflet of the membrane, which incorporate newly externalised precursors into the existing network (vancomycin and penicillin). In B. subtilis, the latter reactions are catalysed along the sidewalls by the Rod complex, thought to primarily build the sacculus, and by class A penicillin-binding proteins (aPBPs), thought to add to repair it. Our findings reveal that the two antibiotic groups lead to growth arrest, sacculus thinning, and eventual cell lysis. However, while the impact of vancomycin and penicillin G is rapid, lacking morphological deformation, fosfomycin and D-cycloserine induce cell widening and bulging before lysis. During shortage of PG precursors, dysregulated PG hydrolytic activity contributes to elevated cell lysis but is not responsible of bulging. Instead, dispersed PG synthesis by aPBPs persists while the activity of the Rod system is rapidly arrested, resulting in cell rounding. We propose that this facilitates the redirection of the limited PG precursors to sites of CW repair, thereby preserving cell integrity and allowing for prolonged growth during antibiotic challenge.

Adherent Invasive Escherichia coli (AIEC) colonize the ileal mucosa of Crohn's disease patients. Among bacterial virulence factors potentially implicated, type 6 secretion systems (T6SS) are molecular machineries sending effectors in prokaryotic or eukaryotic target cells. Two T6SS have been reported in AIEC LF82 reference strain. Here, we show that T6SS genes are more prevalent in Crohn's disease patients' microbiota compared to healthy controls, and that T6SS are more frequent in AIEC pathovar compared to E. coli in general and associated with FimH pathoadaptive polymorphism. The use of an in vitro gastrointestinal system coupled with T6SS promoter reporter assay highlighted expression of LF82 T6SS under gastrointestinal conditions, with a major role for bile salts. In mice infected with AIEC LF82 versus T6SS-deleted mutants, T6SS showed a functional impact in modulating the composition of mucosa-associated gut microbiota. Thus, AIEC may use their T6SS to compete with endogenous microbiota to colonize host mucosa.

The Atc protein system (AtcJ, AtcA, AtcB and AtcC) plays a major role in cold adaptation in Shewanella oneidensis. AtcJ, a J-domain protein, interacts with the DnaK chaperone and binds AtcC through a conserved C-terminal PX7W motif in which Trp is crucial. Overproduced AtcB inhibits RNA polymerase, while the AtcJ-AtcC complex recruits DnaK to modulate this inhibition. This study investigates the conservation of these functional traits across divergent bacteria. We characterized several non-canonical Atc systems and demonstrated that the core interaction network remains functionally preserved despite remarkable variability in the AtcJ C-terminal motif, ranging from intact PX7W to degenerate or absent forms, but also in the size of AtcC. Phylogenetic analysis revealed vertical inheritance and co-evolution with host transcriptional machinery. AtcB, AtcC, and AtcJ exhibit strong phylogenetic congruence with the species tree, while AtcA shows greater evolutionary flexibility. The system is enriched in aquatic and psychrophilic lineages but absent in thermophiles. Conditional toxicity assays revealed that AtcB-RNAP interaction depends on specific structural determinants, with DnaK recruitment alleviating toxicity. These findings support a unified molecular mechanism coupling chaperone activity to transcriptional regulation under environmental stress, suggesting that the Atc system represents an ancient and adaptable regulatory module in bacteria.

The endonuclear symbiont Holospora obtusa invades the macronucleus of Paramecium caudatum using an infectious-form invasion-tip containing a major 89-kDa periplasmic protein implicated in escape from the host digestive vacuole and macronuclear invasion. Sequence-based analyses placed this protein within a Holospora-specific family and predicted an N-terminal membrane-anchoring module and a long alpha-helical C-terminal region. AlphaFold2 modeling followed by molecular dynamics indicated that the C-terminal region forms an antiparallel, spectrin-repeat-like helical solenoid containing a localized three-helix coiled-coil. The protein exhibits a large pH-dependent charge difference, becoming 55 units more positive at pH 4 than at pH 7. Independent simulations at these pH values differed mainly in the relative orientation of the N-terminal core and C-terminal rod, described by DynDom as an approximately 35 degrees rigid-body rotation about a hinge centered on Gly291. However, none of the nearby glutamates (Glu273, Glu275, and Glu302) formed a pH-dependent salt bridge, and no specific protonation-dependent interaction responsible for the conformational difference was identified. We nonetheless propose, as a testable hypothesis, that acidification of the digestive vacuole may promote this reorientation, thereby repositioning the N-terminal membrane-anchoring module at the bacterial surface as an early step toward escape from the vacuole.

Uropathogenic Escherichia coli (UPEC) is the predominant causative agent of urinary tract infections. The UPEC strain, CFT073, has a mosaic genome containing 60 genomic islands enriched in virulence genes. In this study, we explore the small proteome of CFT073 using ribosome profiling (Ribo-seq) coupled with RNA sequencing (RNA-seq), and reveal 338 translated, previously unannotated, putative small open reading frames (sORFs) with less than 100 codons. Among them, 78 sORFs are located in intergenic regions. Fifteen intergenic sORFs encode a predicted transmembrane helix (smemORFs), of which seven are hypothetical or have not been characterized in other E. coli strains. The majority of the remaining putative sORFs encodes uncharacterized or novel soluble proteins (ssolORFs). Considering the evolution of many sORFs is likely to be recent, functional sORFs are more likely to have conserved gene neighborhood in other genomes. Searching into the genomes of 10 other E. coli strains and four enterobacteria species, synteny analysis reveals that 10 of 15 putative smemORFs, and 27 of 63 putative ssolORFs, have a homologous sequence in a similar genomic context in at least one other genome, supporting their likelihood of encoding functional proteins. Further validation of putative sORFs using proteomics, cell-free protein synthesis, and mNeonGreen tagging provide strong evidence for the translation of nine predicted intergenic sORFs. Notably, four previously uncharacterized small membrane proteins have increased abundance under oxidative stress, suggesting a potential role in the stress response. Together, our findings reveal the small proteome of an extraintestinal pathogenic E. coli and provide a catalog of putative sORFs for further investigation.

A subgroup of membrane-anchored bacterial Alkaline Phosphatase superfamily members transfers phospholipid headgroups from phospholipids to diverse acceptor molecules. As part of type II and hybrid type I/II cellulose biosynthesis gene clusters BcsG transfers phosphorylethanolamine pEtN from phospholipid to the emerging 1,4 {beta}-D-glucan chain synthesized by the BcsABC cellulose biosynthesis nanomachine. BcsG homologs throughout the phylogenetic tree possess a uniquely structured tripartite linker that connects the fifth transmembrane domain with the periplasmic catalytic domain. A N-terminal unstructured sequence of highly variable length and biased amino acid composition, the N-linker, is followed by an -helix and an unstructured sequence of high similarity and constant length leading into the first {beta}-strand of the catalytic domain. With BcsG from Salmonella typhimurium synthesizing pEtN cellulose as a model, linkers were shown to be promiscuous and linker length positively correlated with transfer efficiency of the pEtN headgroup and protein stability. As the donor substrate, the phosphatidylethanolamine content of the membranes affects pEtN transfer efficiency. With Alkaline Phosphatase superfamily members to be promiscuous enzymes the catalytic domain of OpgE transfers pEtN to osmoregulated periplasmic glucan. BcsG-OpgE hybrid proteins cause colony morphology alterations thus potentially transferring pEtN to the BcsA synthesized glucan chain. With BcsG with shortest linkers not associated with a cellulose biosynthesis gene cluster, multiple hypotheses can be proposed that select for different length of the N-linker in BcsG homologs located within a cellulose biosynthesis gene cluster in other Pseudomonadati.

To persist during infection Klebsiella pneumoniae must overcome nutrient iron and zinc limitation imposed by the host immune system through a process called nutritional immunity. Secreted small molecule siderophores are a major virulence determinant of Klebsiella pneumoniae pathogenesis and are presumed to overcome nutritional immunity by binding iron for bacterial acquisition. In this work, we set out to identify how a multi-drug resistant K. pneumoniae grows in zinc limited environments. Using unbiased transcriptomics, proteomics, and an arrayed transposon screen, we identified that synthesis and uptake of the siderophore enterobactin is required to allow for growth in low zinc conditions. Iron-specific chelators did not replicate this phenotype and addition of supplemental iron through heme in growth media could not complement severe growth defects of enterobactin mutant K. pneumoniae experiencing zinc limitation. Finally, zinc starvation induced enterobactin production independent of the canonical zinc uptake regulator (Zur) transcription factor suggesting an unidentified regulatory mechanism by which Gram-negative pathogens may respond to zinc stress. Together, these studies expand the role of enterobactin beyond iron regulation and highlight a previously unreported link between iron and zinc homeostasis in Klebsiella pneumoniae.

A substantial fraction of the Mycobacterium tuberculosis proteome remains functionally uncharacterised. Rv1025, a 155-residue protein carrying the domain of unknown function DUF501 (Pfam PF04417), is essential by transposon mutagenesis and vulnerable by CRISPR interference, an attractive but neglected drug target, yet has never been functionally described. The family (4,370 proteins, no Gene Ontology term, no solved structure) is uncharacterised across all organisms and essential in three Actinobacterial genera. A Foldseek search of the AlphaFold model against complete structural databases finds no significant homolog, indicating a novel fold. The operon eno-divIC-Rv1025-ppx2 is conserved across the Actinobacteria phylum, yet AlphaFold-Multimer finds no direct complex between Rv1025 and its neighbour DivIC. Instead, conservation across 8,700 homologous sequences reveals a near-invariant Cys113-His115-Glu59 cluster forming a pocket. Holo AlphaFold3 predictions with Zn, Fe and Mn confidently place a divalent metal on this triad at 2.25-2.47 A; mutating the triad relocates the metal, and an independent backbone-geometry predictor recovers the same site, confirming specificity. The triad is universal across the family: present in all 1,472 near-complete bacterial sequences of the Pfam alignment, with no non-conservative substitution among the 2,228 sequences examined, a defining feature of bacterial DUF501 rather than a mycobacterial peculiarity. We propose that DUF501 is a metal-binding protein and candidate metalloenzyme, the first functional hypothesis for this family, whose conserved, essential metal pocket is a promising drug target. As the predictions build on a conservation-defined site within a fully computational study, they are supportive rather than proof of metal occupancy and warrant experimental validation.

Streptococcus pneumoniae is a major human respiratory pathogen. The bacterial 70S ribosome is a target of many clinically important antibiotics. The N-terminus of ribosomal protein bL27 extends into the peptidyl transferase center and contributes to the translation process. In Firmicutes, full length bL27 contains an 8-12 amino acid N-terminal extension that is absent from Gram-negative bacteria. This extension is cleaved by the protease Prp, which is absent from organisms lacking the extension. Prp-mediated cleavage of bL27 is essential in Staphylococcus aureus, and Prp has been proposed as a potential antibiotic target. Here, we show that in S. pneumoniae strain TIGR4, a {Delta}prp mutant remained viable, and produced ribosomes containing cleaved bL27, whereas deletion of prp was not tolerated in strain D39. These results suggested the presence of an alternate bL27-processing protease in TIGR4 that was absent from D39. Using a combination of genomics, proteomics and biochemical analyses, we identified this enzyme as the product of previously uncharacterized gene SP_1145, encoding a protease that we named Ribosome rescue protease (Rrp). SP_1145 is carried on a mobile genetic element that is present in strain TIGR4, but absent from D39. Our findings shed light on an alternative mechanism for bL27 maturation, and indicate that some strains of S. pneumoniae harbor horizontally acquired redundant pathways for this essential ribosome processing step.

Small annotated open reading frames are the most neglected part of the functionally uncharacterised M. tuberculosis genome. We revisit Rv0810c, a 60-residue protein carrying the unknown-function domain DUF3073 (Pfam PF11273), flagged as an Actinobacteria-signature protein in 2006 but never studied since. Rv0810c is genuinely translated (detected in 11 of 16 M. tuberculosis proteomic datasets), with no significant human homologue, no neighbouring-gene overlap, and no CRISPR-interference polar effect on either flank. Residue-resolved confidence reveals a bipartite architecture: a rigid 33-residue module (pLDDT 91.9) followed by an extended, acidic, intrinsically disordered tail (radius of gyration 22.8 A against 11-12 A expected for a globular protein). The gene is under strong purifying selection (non-synonymous/synonymous ratio 0.86 against 1.93 among 74 size-matched controls, p=5.8x10-8), and its two commonest missense variants are each confined to one sub-lineage, indicating clonal expansion rather than relaxed constraint. DUF3073 is present without a single confirmed loss across 260 well-supported Actinomycetia genera. Despite this conservation, eight independent computational strategies, spanning sequence, structure, electrostatic-patch, embedding-similarity and homo-oligomerisation searches, converge on the same negative: no assignable fold, binding site, or functional neighbour in curated or uncurated sequence space. A phosphosite (Thr24), reproducibly reported by three laboratories, cannot be attributed to a kinase by chemical-genetic or sequence-motif evidence. The contradiction between predicted cytoplasmic topology and macrophage-secretory-fraction detection is narrowed, not resolved: ESX secretion, an immunodominant-epitope confound and host-induced transcription are excluded. Rv0810c exemplifies a class of genuinely uncharacterisable small proteins for which negative reporting, not a manufactured function, is the honest outcome.

Bacterial virulence genes exhibit distinctive codon usage that may impose tRNA-modification requirements for efficient translation, but a comprehensive understanding is lacking. Here, a curated codon-tRNA modification map for Salmonella predicted six modifications that could preferentially support virulence gene translation. Mutant phenotypes in infected mice confirmed requirements for iA37 and C5 modification of U34, whereas essential k2C34 could not be tested. Q34, Cm32/Um32, and Cm34 had limited impact consistent with weak decoding effects in other systems. Non-predicted tRNA modifications were not required. Synonymous reporter variants, proteomics, and targeted recoding demonstrated that virulence-associated codons directly conferred modification dependence. Additionally, four Leu-TTA codons in ssrB, the master regulator of Salmonella pathogenicity island 2 (SPI-2), contributed to MiaA-dependent SPI-2 gene expression. Extending our analysis to other pathogens predicted shared and divergent virulence dependencies consistent with published mutant phenotypes. Thus, codon usage explains tRNA-modification requirements for bacterial virulence.

The genus Mycobacterium comprises over 200 species, many of which now have complete genome sequences. Some are major pathogens causing diseases like tuberculosis and leprosy, while others are harmless environmental organisms with useful abilities such as degrading pollutants. Environmental mycobacteria are often seen as metabolic generalists, able to utilise a wider range of carbon sources than host-associated species, which are typically more specialised due to their restricted habitats. This metabolic versatility has been proposed to stem from differences in nutrient uptake capabilities rather than catabolic pathways. In order to test this explanation, we developed and validated genome-scale metabolic models for five Mycobacterium species with varying lifestyles and growth rates, creating a computational approach enabled by CarveMe that allows rapid construction of models from genome information. By combining these models with microbiology experiments the study showed that the capacity of the bacteria to transport nutrients into the cell is indeed key to metabolic versatility. We notably found through load-partition experiments that, if a transporter is present but cannot take up its substrate at a rate sufficient for growth, the supply of multiple substrates can mitigate this rate-limiting step. This suggests that mycobacterial species have evolved high-affinity, low-rate systems for nutrient uptake in their ecological niches. More generally, our results demonstrate that a combination of automated annotation methods and straightforward bacterial physiology experiments allow the reconstruction of metabolic models of good predictive quality for hitherto little studied mycobacterial species.

Siderophore transport is central to microbial competition, because it determines access to iron, frequently a limiting nutrient. While siderophore-mediated iron uptake via TonB-dependent transporters (TBDTs) has been extensively studied in heterotrophic bacteria, little is known about the functionality and specificity of TBDTs in cyanobacteria. In the present study we functionally characterise the import system of cyanochelin B, a photolytic {beta}-hydroxy aspartate siderophore produced by several filamentous cyanobacteria, including Leptolyngbya sp. NIES-3755. We have identified a cyanochelin B putative transport cassete localized in the vicinity of the cyanochelin biosynthetic gene cluster in Leptolyngbya genome. By expressing the import genes heterologously in a model unicellular cyanobacterium Synechocystis sp. PCC 6803, we established that the transport cassette reconstitutes cyanochelin B-dependent growth, consistent with cyanochelin-mediated iron acquisition. Systematic gene dissection showed that the TBDT (CctA) and the substrate-binding protein (CctB), responsible for binding the siderophore in the periplasm, are alone sufficient for cyanochelin import into Synechocystis cells, with the permeases, ATPase and a cassette-associated ferredoxin supplied in trans by the host. CctA carries an N-terminal AMIN domain, a fusion found only in cyanobacterial TBDTs. The cassette accepts the structurally similar cyanochelin A but not cyanochelin C, enterobactin or pyoverdine, indicating limited promiscuity. The phylogenetic placement of cyanochelin receptors within a broader clade containing citrate-hydroxamate-type siderophore receptors suggests an evolutionary link between transport systems for chemically distinct cyanobacterial siderophores. Our study reports the first functional heterologous expression of a cyanobacterial TonB-dependent transporter and establishes Synechocystis as a promising platform for cyanobacterial xenosiderophore-uptake studies.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes acute and chronic opportunistic infections. Progression to chronic infection requires upregulation of the Rsm small regulatory RNAs (sRNAs), which in turn induce the expression of virulence factors to promote chronic virulence phenotypes, including biofilm formation. Iron is a critical nutrient for survival and virulence of Pa, and host-mediated iron limitation leads to the induction of key virulence factors driving infection. Prior work showed that the RsmY and RsmZ sRNAs are modestly upregulated due to iron limitation in static growth conditions via PqsR. Here, we show that another Rsm sRNA, RsmW, is strongly induced by iron limitation, likely in a Fur-dependent manner. We identified an RNA processing event, which appears to be mediated by the RNAse E, resulting in cleavage of RsmW from the 3 UTR of pa4570 mRNA. We also report that RsmW is more stable than the full length pa4570-rsmW transcript as well as the processed pa4570 mRNA. Surprisingly, we found that RetS, which down-regulates RsmY and RsmZ expression, enhances expression of RsmW under iron limiting conditions. We further demonstrate that RsmW plays a key role in biofilm formation under iron limiting conditions and is required for full pathogenesis in a Galleria mellonella infection model. Lastly, we observed similar iron-dependent regulation of RsmW in chronic infection isolates from cystic fibrosis (CF) sputum, underlining the potential importance of this regulation in the CF lung. Taken together, the results outline a new paradigm for sRNA-dependent iron regulation of P. aeruginosa pathogenesis.

Shigella flexneri is a leading cause of diarrheal disease worldwide, yet bacterial factors required within distinct host niches remain poorly understood. Here, we used genome-wide transposon sequencing in an infant rabbit model of shigellosis to identify genes promoting bacterial fitness in vivo. The screen identified virulence factors on the large virulence plasmid and novel chromosomal fitness factors. Characterization of zitB, encoding a cation diffusion facilitator family Zn transporter, revealed a niche-specific fitness contribution. The {Delta}zitB mutant showed no growth defect in vitro in rich media or in vivo during the early infection phase (8 hpi) in epithelial cells. However, bacterial burden was reduced during the late infection phase (24 hpi) in vivo, when bacteria interact with immune cells, in both competitive and mono-infection assays. Reduced bacterial burden was associated with increased MARCO-specific macrophages, suggesting impaired colonization and killing by the {Delta}zitB mutant. Consistently, {Delta}zitB fitness was impaired in THP-1-derived macrophages. Metal chelation increased bacterial burden, indicating a role for macrophage-mediated metal toxicity. The {Delta}zitB mutant also showed increased sensitivity to zinc and copper. Finally, macrophage depletion in vivo restored bacterial burden to wild-type levels. Together, these findings identify ZitB as a niche-specific bacterial factor promoting S. flexneri fitness during macrophage-associated metal stress.

Bacterial cultures entering stationary phase (SP) undergo complex physiological alterations, helping the cells to survive when medium resources are exhausted. The onset of various stress responses underlying SP physiology is generally believed to be due to reactions of individual cells to the environmental cues such as starvation, toxic metabolites, etc. The SP physiological state makes bacteria unsuitable for the replication of most bacteriophages; however, some phages are able to infect and multiply in them. We investigated Escherichia coli phage DH23 growth in SP cultures of E. coli MG1655 infected at different hours post inoculation (ages). Under our conditions the cells enter SP at about 6 h, but the phage replication was possible till 11h and then dropped abruptly by 13h of culture aging, indicating an abrupt physiological change to deeper dormancy during SP. The onset of this phage-inhibiting middle-SP dormancy (MSPD) turned out to be mediated by intercellular communication mediated by the middle-SP signal particles (MSP) which are larger than 100 kDa and contain both RNA and DNA. These MSP are inactivated by RNAse or by DNAse, enabling phage growth in 14h-old cultures and lifts the tolerance of such cultures to kanamycin. Moreover, the RNAse or DNAse treatment makes 24h culture spent media suitable for additional cell growth, and cultures initiated in fresh LB supplemented with RNAse reach an OD600 about 1.5 times higher compared to the untreated control. This indicates that MSP signaling influences cell physiology well before the MSPD onset and helps the population cease growth pre-emptively to save about 1/3 of medium resources to support the viability during SP.