The 50 most-cited articles in Journal of Bacteriology, 2019

Lag is a temporary period of nonreplication seen in bacteria that are introduced to new media. Despite latency being described by Müller in 1895, only recently have we gained insights into the cellular processes characterizing lag phase. This review covers literature to date on the transcriptomic, proteomic, metabolomic, physiological, biochemical, and evolutionary features of prokaryotic lag. Though lag is commonly described as a preparative phase that allows bacteria to harvest nutrients and adapt to new environments, the implications of recent studies indicate that a refinement of this view is well deserved. As shown, lag is a dynamic, organized, adaptive, and evolvable process that protects bacteria from threats, promotes reproductive fitness, and is broadly relevant to the study of bacterial evolution, host-pathogen interactions, antibiotic tolerance, environmental biology, molecular microbiology, and food safety.

and other bacteria. This phenomenon is of interest for understanding bacterial physiology but is also of practical relevance. Growth inhibition by organic acids underlies food preservation and causes problems during high-density fermentation in biotechnology. What causes this phenomenon? Classical explanations invoke the uncoupling effect of acetate and the establishment of an anion imbalance. Here, we propose and investigate an alternative hypothesis: the perturbation of acetate metabolism due to the inflow of excess acetate. We find that this perturbation accounts for 20% of the growth-inhibitory effect through a modification of the acetyl phosphate concentration. Moreover, we argue that our observations are not expected based on uncoupling alone.

In this review, we discuss the influence of polymicrobial interactions on the antibiotic susceptibility of biofilms, and we highlight the studies that first documented the shifted antimicrobial susceptibilities of mixed-species cultures. Recent studies have identified several mechanisms underlying the recalcitrance of polymicrobial biofilm communities, including interspecies exchange of antibiotic resistance genes, β-lactamase-mediated inactivation of antibiotics, changes in gene expression induced by metabolites and quorum sensing signals, inhibition of the electron transport chain, and changes in properties of the cell membrane. In addition to elucidating multiple mechanisms that contribute to the altered drug susceptibility of polymicrobial biofilms, these studies have uncovered novel ways in which polymicrobial interactions can impact microbial physiology. The diversity of findings discussed highlights the importance of continuing to investigate the efficacy of antibiotics against biofilm communities composed of different combinations of microbial species. Together, the data…

The most common intracellular symbiont on the planet— Wolbachia pipientis —is infamous largely for the reproductive manipulations induced in its host. However, more recent evidence suggests that this bacterium may also serve as a nutritional mutualist in certain host backgrounds and for certain metabolites. We performed a large-scale analysis of conserved gene content across all sequenced Wolbachia genomes to infer potential nutrients made by these symbionts.

This study describes the characterization of nisin J, the first example of a natural nisin variant, produced by a human skin isolate of staphylococcal origin. Nisin J displays inhibitory activity against a wide range of bacterial targets, including MRSA. This work demonstrates the potential of human commensals as a source for novel antimicrobials that could form part of the solution to antibiotic resistance across a broad range of bacterial pathogens.

Bacteria in biofilms are protected from killing by antibiotics, and this reduced susceptibility contributes to the persistence of infections such as those in the cystic fibrosis lung and chronic wounds. A generalized conceptual model of biofilm antimicrobial tolerance with the following mechanistic steps is proposed: (i) establishment of concentration gradients in metabolic substrates and products; (ii) active biological responses to these changes in the local chemical microenvironment; (iii) entry of biofilm cells into a spectrum of states involving alternative metabolisms, stress responses, slow growth, cessation of growth, or dormancy (all prior to antibiotic treatment); (iv) adaptive responses to antibiotic exposure; and (v) reduced susceptibility of microbial cells to antimicrobial challenges in some of the physiological states accessed through these changes.

ABSTRACT Iron is a critical nutrient for most microbial pathogens, and the innate immune system exploits this requirement by sequestering iron and other metals through a process termed nutritional immunity. The opportunistic pathogen Pseudomonas aeruginosa provides a model system for understanding the microbial response to host iron depletion, as this organism exhibits a high requirement for iron as well as an exquisite ability to overcome iron deprivation during infection. Hallmarks of P. aeruginosa’s iron starvation response include the induction of multiple high affinity iron acquisition systems and an “iron sparing response” that is post-transcriptionally mediated by the PrrF small regulatory RNAs (sRNAs). Here, we used liquid chromatography-tandem mass spectrometry to conduct label-free proteomics of the P. aeruginosa iron starvation response, revealing several iron-regulated processes that have not been previously described. Iron starvation induced multiple proteins involved in branched chain and aromatic amino acid catabolism, providing the capacity for iron-independent entry…

We uncover a phage- and antibiotic-induced stress response in the clinically important opportunistic pathogen Pseudomonas aeruginosa . Phage-infected P. aeruginosa subpopulations are isolated from uninfected subpopulations by the production of a stress-induced signal. Activation of the stress response by antibiotics causes P. aeruginosa to physically be repelled from the area containing antibiotics altogether, consistent with a mechanism of antibiotic evasion. The stress response observed here could increase P. aeruginosa resilience against antibiotic treatment and phage therapy in health care settings, as well as provide a simple evolutionary strategy to avoid areas containing stress.

There is growing evidence for a link between gastrointestinal bacterial communities and airway disease progression in CF. We demonstrate that infants with CF ≤1 year of age show a distinct stool microbiota versus that of control infants of a comparable age. We detected associations between the gut microbiome and airway exacerbation events in the cohort of infants with CF, andin vitrostudies provided one possible mechanism for this observation. These data clarify that current therapeutics do not establish in infants with CF a gastrointestinal microbiota like that in healthy infants, and we suggest that interventions that direct the gastrointestinal microbiota closer to a healthy state may provide systemic benefits to these patients during a critical window of immune programming that might have implications for lifelong health.

Abstract Flagellar motility is critical for surface attachment and biofilm formation in many bacteria. A key regulator of flagellar motility in Pseudomonas aeruginosa and other microbes is cyclic diguanylate (c-di-GMP). High levels of this second messenger repress motility and stimulate biofilm formation. C-di-GMP levels regulate motility in P. aeruginosa in part by influencing the localization of its two flagellar stator sets, MotAB and MotCD. Here we show that just as c-di-GMP can influence the stators, stators can impact c-di-GMP levels. We demonstrate that the swarming motility-driving stator MotC physically interacts with the transmembrane region of the diguanylate cyclase SadC. Furthermore, we demonstrate that this interaction is capable of stimulating SadC activity. We propose a model by which the MotCD stator set interacts with SadC to stimulate c-di-GMP production in conditions not permissive to motility. This regulation implies a positive feedback loop in which c-di-GMP signaling events cause MotCD stators to disengage from the motor; then disengaged stators stimulate…

The finding that exposure to DNase I impairs biofilm formation or leads to the dispersal of early stage biofilms has led to the realization of extracellular genomic DNA (eDNA) as a structural component of the biofilm matrix. However, little is known about the contribution of intrinsic DNases to the weakening of the biofilm matrix and dispersion of established biofilms. Here, we demonstrate for the first time that nucleases are induced in dispersed Pseudomonas aeruginosa cells and are essential to the dispersion response and that degradation of matrix eDNA by endogenously produced/secreted EndA is required for P. aeruginosa biofilm dispersion. Our findings suggest that dispersing cells mediate their active release from the biofilm matrix via the induction of nucleases.

Abstract Here we introduce plasmids for xylose-regulated expression and repression of genes in Clostridioides difficile . The xylose-inducible expression vector allows for ~100-fold induction of an mCherryOpt reporter gene. Induction is titratable and uniform from cell-to-cell. The gene repression plasmid is a CRISPR-interference (CRISPRi) system based on a nuclease-defective, codon-optimized allele of the Streptococcus pyogenes Cas9 protein ( dCas9 ) that is targeted to a gene of interest by a constitutively-expressed single guide RNA (sgRNA). Expression of dCas9 is induced by xylose, allowing investigators to control the timing and extent of gene-silencing, as demonstrated here by dose-dependent repression of a chromosomal gene for a red fluorescent protein (maximum repression ~100-fold). To validate the utility of CRISPRi for deciphering gene function in C. difficile , we knocked-down expression of three genes involved in biogenesis of the cell envelope: the cell division gene ftsZ , the S-layer protein gene slpA and the peptidoglycan synthase gene pbp-0712 . CRISPRi confirmed…

Topological entanglement between daughter chromosomes has to be reduced to exactly zero every time an E. coli cell divides. The enzymatic agents that accomplish this task are the topoisomerases. E. coli possesses four topoisomerases. It has been thought that topoisomerase IV is primarily responsible for unlinking the daughter chromosomes during DNA replication. We show here that topoisomerase III also plays a role in this process and is specifically localized to the replisome, the multiprotein machine that duplicates the cell’s genome, in order to do so.

, which serve as model organisms for Gram-negative and Gram-positive bacteria, respectively. Yet our understanding of bacterial cell division and morphology regulation is far from complete, especially in noncanonical and non-rod-shaped organisms. In this review, we focus on two proteins that are highly conserved in Gram-positive organisms, DivIVA and its homolog GpsB, and attempt to summarize the recent advances in this area of research and discuss their various roles in cell division, cell growth, and chromosome segregation in addition to their interactome and posttranslational regulation.

Selenium is an essential element for life, with Se(IV) reduction a key step in its biogeochemical cycle. This report identifies for the first time a dissimilatory Se(IV) reductase, Srr, from a known selenite-respiring bacterium, the haloalkalophilic Bacillus selenitireducens strain MLS10. The work extends the versatility of the complex iron-sulfur molybdoenzyme (CISM) superfamily in electron transfer involving chalcogen substrates with different redox potentials. Further, it underscores the importance of biochemical and enzymological approaches in establishing the functionality of these enzymes.

Bacteria rely on a sophisticated envelope to shield them against challenging environmental conditions and therefore need to ensure correct envelope assembly and integrity. A major signaling pathway that performs this role in Gram-negative species is the Cpx system. An outer membrane lipoprotein of unclear function, NlpE, has long been exploited as a research tool to study Cpx in E. coli , since it triggers this system when overproduced or mislocalized; however, the mechanism and physiological relevance of the NlpE-Cpx connection have awaited further investigation. We elucidate a new function for NlpE by showing that it physically interacts with the Cpx sensor CpxA and acts as a sentinel that specifically monitors two essential envelope biogenesis processes, namely, lipoprotein sorting and oxidative folding.

Potassium is an essential ion in every living cell. Even though potassium is the most abundant cation in cells, its accumulation can be toxic. Therefore, the level of potassium has to be tightly controlled. In many Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in the control of potassium homeostasis by binding to potassium transporters and regulatory proteins and RNA molecules. In the lactic acid bacterium Lactococcus lactis , none of these conserved c-di-AMP-responsive molecules are present. In this study, we demonstrate that the KupA and KupB proteins of L. lactis IL1403 are high-affinity potassium transporters and that their transport activity is inhibited by the second messenger c-di-AMP.

Human gut Bacteroides species exhibit strain-level differences in their physiology, ecology, and impact on human health and disease. However, existing approaches for genetic manipulation generally require construction of genetically modified parental strains for each microbe of interest or defined medium formulations. In this report, we introduce a robust and efficient strategy for targeted genetic manipulation of diverse wild-type Bacteroides species from the human gut. This system enables genetic investigation of members of human and animal microbiomes beyond existing model organisms.

ABSTRACT Competence for genetic transformation allows the opportunistic human pathogen Streptococcus pneumoniae to take up exogenous DNA for incorporation into its own genome. This ability may account for the extraordinary genomic plasticity of this bacterium, leading to antigenic variation, vaccine escape, and the spread of antibiotic resistance markers. The competence system has been thoroughly studied and its regulation is well-understood. Additionally, over the last decade, several stress factors have been shown to trigger the competent state, leading to the activation of several stress response regulons. The arrival of next-generation sequencing techniques allowed us to update the competence regulon, the latest report of which still depended on DNA microarray technology. Enabled by the availability of an up-to-date genome annotation, including transcript boundaries, we assayed time-dependent expression of all annotated features in response to competence induction, were able to identify the affected promoters and produced a more complete overview of the various regulons…

In the environment, many microbes form biofilms. However, the interspecies interactions underlying bacterial coexistence within these biofilms remain understudied. Here, we mimic environmentally relevant biofilms by studying a dual-species biofilm formed between Bacillus subtilis and Pantoea agglomerans and subjecting the coculture to chemical and physical stressors that it may experience in the natural world. We determined that both bacteria contribute structural elements to the coculture, which is reflected in its overall viscoelastic behavior. Existence within the coculture can be either beneficial or detrimental depending on the context. Many of the features and determinants of the coculture biofilm appear distinct from those identified in monoculture biofilm studies, highlighting the importance of characterizing multispecies consortia to understand naturally occurring bacterial interactions.

Pseudomonas aeruginosa forms multicellular aggregates or biofilms encased in a matrix. We show for the first time here that dispersion by P. aeruginosa requires the endogenous expression of pelA and pslG , leading to the degradation of both Pel and Psl polysaccharides, with PslG-induced dispersion being CdrA dependent. The findings suggested that endogenously induced Psl degradation is a sequential process, initiated by untethering of CdrA-bound Psl or CdrA-dependent cell interactions to enable Psl degradation and ultimately, dispersion. Untethering likely involves CdrA release in a manner independent of c-di-GMP modulation and thus LapG. Our findings not only provide insight into matrix degrading factors contributing to dispersion but also identify key steps in the degradation of structural components of the P. aeruginosa biofilm matrix.

ABSTRACT Cystic fibrosis (CF) patients chronically infected with both Pseudomonas aeruginosa and Staphylococcus aureus have worse health outcomes than patients who are mono-infected with either P. aeruginosa or S. aureus . We showed previously that mucoid strains of P. aeruginosa can co-exist with S. aureus in vitro due to transcriptional downregulation of several toxic exoproducts typically produced by P. aeruginosa , including siderophores, rhamnolipids, and HQNO (2-heptyl-4-hydroxyquinoline N-oxide). Here we demonstrate that exogenous alginate protects S. aureus from P. aeruginosa in both planktonic and biofilm co-culture models under a variety of nutritional conditions. S. aureus protection in the presence of exogenous alginate is due to transcriptional downregulation of pvdA , a gene required for the production of the iron scavenging siderophore pyoverdine, as well as down-regulation of the PQS (Pseudomonas quinolone signal; 2-heptyl-3,4-dihydroxyquinoline) quorum sensing system. The impact of exogenous alginate is independent of endogenous alginate production. We further…

and that most bacteria enforce the DNA damage checkpoint by expressing a small integral membrane protein. Resumption of cell division is then mediated by membrane-bound proteases that cleave the cell division inhibitor. Further, many bacterial cells have mechanisms to inhibit cell division that are regulated independently from the canonical LexA-mediated SOS response. In this review, we discuss several pathways used by bacteria to prevent cell division from occurring when genome instability is detected or before the chromosome has been fully replicated and segregated.

The ability of Pseudomonas aeruginosa to form biofilms, which are communities of cells encased in a self-produced extracellular matrix, protects the cells from antibiotics and the host immune response. While some biofilm matrix components, such as exopolysaccharides and extracellular DNA, are relatively well characterized, the extracellular matrix proteins remain understudied.

Bacteriocins (ribosomally produced antimicrobial peptides) are potential alternatives to current antimicrobials given the global challenge of antimicrobial resistance. We identified a novel bacteriocin from Actinomyces ruminicola with no previously characterized antimicrobial activity. Using publicly available genomic data, we found a highly conserved yet divergent family of previously unidentified homologous peptide sequences within the genus Actinomyces with striking similarity to eukaryotic defensins. These actifensins may provide a potent line of antimicrobial defense/offense, and the machinery to produce them could be used for the design of new antimicrobials given the degeneracy that exists naturally in their structure.

Type III secretion systems (T3SS) are widely distributed in Gram-negative microorganisms and critical for host-pathogen and host-symbiont interactions with plants and animals. Central features of the T3SS are a highly conserved set of secretion and translocation genes and contact dependence wherein host-pathogen interactions trigger effector protein delivery and serve as an inducing signal for T3SS gene expression.

CRISPR-Cas systems incorporate short DNA fragments from invasive genetic elements into host CRISPR arrays in order to generate host immunity.Recently, we demonstrated that the Csa3a regulator protein triggers CCN PAM-dependent CRISPR spacer acquisition in the subtype I-A CRISPR-Cas system of Sulfolobus islandicus. However, the mechanisms underlying specific protospacer selection and spacer insertion remained unclear. Here, we demonstrate that two Cas4 family proteins (Cas4 and Csa1) have essential roles (a) in recognizing the 5' PAM and 3' nucleotide motif of protospacers and (b) in determining both the spacer length and its orientation. Furthermore, we identify uncovered amino acid residues of the Cas4 proteins that facilitate these functions. Overexpression of the Cas4 and Csa1 proteins, and also of an archaeal virus-encoded Cas4 protein, resulted in strongly reduced adaptation efficiency and the formers yielded a high incidence of PAM-dependent atypical spacer integration, or of PAM-independent spacer integration. We further demonstrated that, in the plasmid challenging…

While many posttranscriptional modifications in M. jannaschii tRNAs are also found in bacteria and eukaryotes, several that are unique to archaea were identified. By RNA modification mapping, the modification profiles of M. jannaschii tRNA anticodon loops were characterized, allowing a comparative analysis with H. volcanii modification profiles as well as a general comparison with bacterial and eukaryotic decoding strategies. This general comparison reveals that M. jannaschii , like H. volcanii , follows codon-decoding strategies similar to those used by bacteria, although position 37 appears to be modified to a greater extent than seen in H. volcanii .

Biofilms occur in a broad range of environments under heterogeneous physicochemical conditions, such as in bioremediation plants, on surfaces of biomedical implants, and in the lungs of cystic fibrosis patients. In these scenarios, biofilms are subjected to shear forces, but the mechanical integrity of these aggregates often prevents their disruption or dispersal. Biofilms' physical robustness is the result of the multiple biopolymers secreted by constituent microbial cells which are also responsible for numerous biological functions. A better understanding of the role of these biopolymers and their response to dynamic forces is therefore crucial for understanding the interplay between biofilm structure and function. In this paper, we review experimental techniques in rheology, which help quantify the viscoelasticity of biofilms, and modeling approaches from soft matter physics that can assist our understanding of the rheological properties. We describe how these methods could be combined with synthetic biology approaches to control and investigate the effects of secreted polymers…

Fusobacterium spp. are emerging pathogens that contribute to mammalian and human diseases, including colorectal cancer. Despite a validated connection with disease, few proteins have been characterized that define a direct molecular mechanism for Fusobacterium pathogenesis. We report a comprehensive examination of virulence-associated protein families in multiple Fusobacterium species and show that complete genomes facilitate the correction and identification of multiple, large type 5a secreted autotransporter genes in previously misannotated or fragmented genomes. In addition, we use protein sequence similarity networks and human cell interaction experiments to show that previously predicted noninvasive strains can indeed bind to and potentially invade human cells and that this could be due to the expansion of specific virulence proteins that drive Fusobacterium infections and disease.

Some bacteria form flocs, which are multicellular floating assemblages of many thousands of cells. Flocs have been relatively little studied compared to surface-adherent biofilms, but flocculation could play many physiological roles, be a crucial factor in marine carbon burial, and enable more efficient biotechnological cell harvesting. We studied floc formation and architecture in the model cyanobacterium Synechocystis sp. strain PCC 6803, using mutants to identify specific cell surface structures required for floc formation. We show that floc formation is regulated by blue and green light perceived by the photoreceptor Cph2. The flocs have a characteristic structure based on strands of linked cells aggregating into dense clusters. Cells within the dense clusters show signs of nutrient stress, pointing to a disadvantage of floc formation.

Production of polyP in bacteria is required for virulence and stress response, but little is known about how bacteria regulate polyP levels in response to changes in their environments. Understanding this regulation is important for understanding how pathogenic microbes resist killing by disinfectants, antibiotics, and the immune system. In this work, I have clarified the connections between polyP regulation and the stringent response to starvation stress in Escherichia coli and demonstrated an important and previously unknown role for the transcription factor DksA in controlling polyP levels.

Lanthanide metals, or rare earth elements, are abundant in nature and used heavily in technological devices. Biological interactions with lanthanides are just beginning to be unraveled. Until very recently, microbial mechanisms of lanthanide metal interaction and uptake were unknown. The TonB-dependent receptor LanA is the first lanthanum receptor identified in a methanotroph. Sequence homology searches with known metal transporters and regulators could not be used to identify LanA or other lanthanide metal switch components, and this method for mutagenesis and selection was required to identify the receptor. This work advances the knowledge of microbe-metal interactions in environmental niches that impact atmospheric methane levels and are thus relevant to climate change.

The bacterial flagellum has evolved as one of the most sophisticated self-assembled molecular machines, which confers locomotion and is often associated with virulence of bacterial pathogens. Variation in species-specific features of the flagellum, as well as in flagellar number and placement, results in structurally distinct flagella that appear to be adapted to the specific environments that bacteria encounter. Here, we used cutting-edge imaging techniques to determine high-resolution in situ structures of polar flagella in Pseudomonas aeruginosa and peritrichous flagella in Salmonella enterica serovar Typhimurium, demonstrating substantial variation between flagella in these organisms. Importantly, we observed novel flagellar subassemblies and provided additional insight into the structural basis of flagellar assembly and loss in both P. aeruginosa and S . Typhimurium.

Abstract Pathogens express a set of proteins required for establishing and maintaining an infection, termed virulence life-style genes (VLGs). Due to their outsized importance in pathogenesis, VLG products are attractive targets for the next generation of antimicrobials. However, precise manipulation of VLG expression in the context of infection is technically challenging, limiting our ability to understand the roles of VLGs in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of gene knockdown tools that are transferred by conjugation and stably integrate into pathogen genomes that we call “Mobile-CRISPRi”. Here we show the efficacy of Mobile-CRISPRi in controlling VLG expression in a murine infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid non-specific toxicity. As a proof of principle, we demonstrate that knockdown of a VLG encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture…

ABSTRACT Extracellular electron transfer (EET), the reduction of compounds that shuttle electrons to distal oxidants, can support bacterial survival when preferred oxidants are not directly accessible. EET has been shown to contribute to virulence in some pathogenic organisms and is required for current generation in mediator-based fuel cells. In several species, components of the electron transport chain (ETC) have been implicated in electron shuttle reduction, raising the question of how shuttling-based metabolism is integrated with primary routes of metabolic electron flow. The clinically relevant bacterium Pseudomonas aeruginosa can utilize carbon sources (i.e., electron donors) covering a broad range of reducing potentials and possesses a branched ETC that can be modulated to optimize respiratory efficiency. It also produces electron shuttles called phenazines that facilitate intracellular redox balancing, increasing the complexity of its metabolic potential. In this study, we investigated the reciprocal influence of respiratory metabolism and phenazine-associated physiology in…

ABSTRACT Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. Here we report that Escherichia coli BL21(DE3), a strain routinely used to overexpress [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an inframe fusion of sufA and sufB genes within the sufABCDSE operon. We show that this fusion protein accumulates in cells but is - inactive in [Fe-S] biogenesis. Restoration of an intact Suf pathway combined with enhanced suf operon expression led to a remarkable (~3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxido-reductase complex. These results show that this engineered ‘SufFeScient’ derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein. IMPORTANCE…

Contact-dependent growth inhibition (CDI) systems are used in bacterial competition to hinder the growth of neighboring microbes. These systems utilize a two-partner secretion mechanism to display the CdiA exoprotein at the bacterial cell surface.

Foliar plant pathogens, like Pseudomonas syringae , adjust their physiology and behavior to facilitate host colonization and disease, but the full extent of these adaptations is not known. Plant immune systems are triggered by bacterial molecules, such as the proteins that make up flagellar filaments. In this study, we found that during plant infection, AlgU, a gene expression regulator that is responsive to external stimuli, downregulates expression of fliC , which encodes the flagellin protein, a strong elicitor of plant immune systems. This change in gene expression and resultant change in behavior correlate with reduced plant immune activation and improved P. syringae plant colonization. The results of this study demonstrate the proximate and ultimate causes of flagellar regulation in a plant-pathogen interaction.

Abstract The Mitis group streptococci include the major human pathogen Streptococcus pneumoniae and the opportunistic pathogens S. mitis and S. oralis which are human oral cavity colonizers and agents of bacteremia and infective endocarditis in immunocompromised patients. Bacterial membrane lipids play crucial roles in microbe-host interactions, yet for many pathogens, the composition of the membrane is poorly understood. In this study, we characterized the lipidomes of selected species of Mitis group streptococci and investigated the mechanistic basis for biosynthesis of the phospholipid phosphatidylcholine (PC). PC is a major lipid in eukaryotic cellular membranes, but it is considered to be comparatively rare in bacterial taxa. Using liquid chromatography/mass spectrometry (LC/MS) in conjunction with stable isotope tracing, we determined that Mitis group streptococci synthesize PC via the rare host metabolite scavenging pathway, the glycerophosphocholine (GPC) pathway, which is largely uncharacterized in bacteria. Our work demonstrates that Mitis group streptococci including S.…

IncA and IncC conjugative plasmids drive antibiotic resistance dissemination among several pathogenic species of Gammaproteobacteria due to the diversity of drug resistance genes that they carry and their ability to mobilize antibiotic resistance-conferring genomic islands such as SGI1 of Salmonella enterica . While historically grouped as “IncA/C,” IncA and IncC replicons were recently confirmed to be compatible and to abolish each other’s entry into the cell in which they reside during conjugative transfer. The significance of our study is in identifying an entry exclusion system that is shared by IncA and IncC plasmids. It impedes DNA transfer to recipient cells bearing a plasmid of either incompatibility group. The entry exclusion protein of this system is unrelated to any other known entry exclusion proteins.

ABSTRACT Lysine acetylation is thought to provide a mechanism for regulating metabolism in diverse bacteria. Indeed, many studies have shown that the majority of enzymes involved in central metabolism are acetylated and that acetylation can alter enzyme activity. However, the details regarding this regulatory mechanism are still unclear, specifically with regards to the signals that induce lysine acetylation. To better understand this global regulatory mechanism, we profiled changes in lysine acetylation during growth of Escherichia coli on the hexose glucose or the pentose xylose at both high and low sugar concentrations using label-free mass spectrometry. The goal was to see whether lysine acetylation differed during growth on these two different sugars. No significant differences, however, were observed. Rather, the initial sugar concentration was the principal factor governing changes in lysine acetylation, with higher sugar concentrations causing more acetylation. These results suggest that acetylation does not target specific metabolic pathways but rather simply targets…

This study shows the involvement of the msaABCR operon in resisting oxidative stress by Staphylococcus aureus generated under in vitro and ex vivo conditions. We show that MsaB regulates the expression and production of a carotenoid pigment, staphyloxanthin, which is a potent antioxidant in S. aureus . We also demonstrate that MsaB regulates the ohr gene, which is involved in defending against oxidative stress generated by organic hydroperoxides. This study highlights the importance of msaABCR in the survival of S. aureus in the presence of various environmental stimuli that mainly exert oxidative stress. The findings from this study indicate the possibility that msaABCR is involved in the persistence of staphylococcal infections and therefore could be a potential antimicrobial target to overcome recalcitrant staphylococcal infections.

In studies of the minimal genetic requirements for life, we used global transposon mutagenesis to identify genes needed for a minimal bacterial genome. Transposon insertion can disrupt the function of a gene but can also have polar effects on the expression of adjacent genes. In the Tn 5 -Puro r construct used in our studies, read-through transcription from Tn 5 -Puro r can drive expression of downstream genes. This results in a preference for Tn 5 -Puro r insertions transcribed toward a downstream essential or quasi-essential gene within the same operon. Such polar effects can have an impact on the classification of genes as essential, quasi-essential, or nonessential, but this has been observed in only a few cases. Also, polar effects of Tn 5 -Puro r insertion can sometimes give clues to gene function.

infections. Collectively, these studies indicate the necessity for further investigation into the role of streptococci in the CF airway to determine how these microbes, alone or via interactions with other CF-associated pathogens, might influence CF lung disease, for better or for worse. We also propose that the interactions of streptococci with other CF pathogens is an ideal model to study clinically relevant microbial interactions.

Salmonella is one of the most important food-borne pathogens, infecting over one million people in the United States every year. These bacteria use a needle-like device to interact with intestinal epithelial cells, leading to invasion of the cells and induction of inflammatory diarrhea. A complex regulatory network controls expression of the invasion system in response to numerous environmental signals. Here we explore the molecular mechanisms by which the small RNA PinT contributes to this regulation, facilitating inactivation of the system after invasion. PinT controls several important virulence systems in Salmonella , tuning the transition between different stages of infection.

Streptococcus gallolyticus is an opportunistic pathogen responsible for septicemia and endocarditis in the elderly and is also strongly associated with colorectal cancer. S. gallolyticus can form biofilms, express specific pili to colonize the host tissues, and produce a specific bacteriocin allowing killing of commensal bacteria in the murine colon. Nevertheless, how the expression of these colonization factors is regulated remains largely unknown. Here, we show that c-di-AMP plays pleiotropic roles in S. gallolyticus , controlling the tolerance to osmotic stress, cell size, biofilm formation on abiotic surfaces, adherence and cell aggregation on human intestinal cells, expression of Pil3 pilus, and production of bacteriocin. This study indicates that c-di-AMP may constitute a key regulatory molecule for S. gallolyticus host colonization and pathogenesis.

ABSTRACT The widespread bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a variety of processes, including protein secretion, motility, cell development, and biofilm formation. C-di-GMP-dependent responses are often mediated by its binding to the cytoplasmic receptors that contain the PilZ domain. We present here comparative structural and sequence analysis of various PilZ-related domains and describe three principal types of them: (i) the canonical PilZ domain, whose structure includes a six-stranded beta-barrel and a C-terminal alpha-helix; (ii) an atypical PilZ domain that contains two extra alpha-helices and forms stable tetramers, and (iii) divergent PilZ-related domains, which include the eponymous PilZ protein and PilZN (YcgR_N) and PilZNR (YcgR_2) domains. We refine the second c-di-GMP binding motif of PilZ as [D/N]hSxxG and show that the hydrophobic residue h of this motif interacts with a cluster of conserved hydrophobic residues, helping maintain the PilZ domain fold. We describe several novel PilZN-type domains that are fused to the canonical PilZ…

Abstract Bacterial genomes harbor cryptic prophages that have lost genes required for induction, excision from host chromosomes, or production of phage progeny. Escherichia coli K12 strains contain a cryptic prophage Qin that encodes a small RNA, DicF, and small protein, DicB, that have been implicated in control of bacterial metabolism and cell division. Since DicB and DicF are encoded in the Qin immunity region, we tested whether these gene products could protect the E. coli host from bacteriophage infection. Transient expression of the dicBF operon yielded cells that were ~100-fold more resistant to infection by λ phage than control cells, and the phenotype was DicB-dependent. DicB specifically inhibited infection by λ and other phages that use ManYZ membrane proteins for cytoplasmic entry of phage DNA. In addition to blocking ManYZ-dependent phage infection, DicB also inhibited the canonical sugar transport activity of ManYZ. Previous studies demonstrated that DicB interacts with MinC, an FtsZ polymerization inhibitor, causing MinC localization to mid-cell and preventing Z ring…

is composed of bioproducers, which are important for the industrial production of chemicals, as well as pathogens, which are a significant burden to the patients and on the health care industry. Historically, even though these bacteria are well known and are commonly studied, the genetic technologies to advance our understanding of these microbes have lagged behind other systems. New tools would continue the advancement of our understanding of clostridial physiology. The genetic modification systems available in several clostridia are not as refined as in other organisms and each exhibit their own drawbacks. With the advent of the repurposing of the CRISPR-Cas systems for genetic modification, the tools available for clostridia have improved significantly over the past four years. Several CRISPR-Cas systems such as using wild-type Cas9, Cas9n, dCas9/CRISPR interference (CRISPRi) and a newly studied Cpf1/Cas12a, are reported. These have the potential to greatly advance the study of clostridial species leading to future therapies or the enhanced production of industrially relevant…