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Control of infections caused by carbapenem-resistant Klebsiella pneumoniae continues to be challenging. The success of this pathogen is favored by its ability to acquire antimicrobial resistance and to spread and persist in both the environment and in humans.The emergence of clinically important clones, such as sequence types 11, 15, 101, and 258, has been reported worldwide. However,the mechanisms promoting the dissemination of such highrisk clones are unknown. Unraveling the factors that play a role in the pathobiology and epidemicity of K. pneumoniae is therefore important for managing infections. To address this issue, we studied a carbapenem-resistant ST-15 K. pneumoniae isolate (Kp3380) that displayed a remarkable adherent phenotype with abundant pilus-like structures. Genome sequencing enabled us to identify a chaperone-usher pili system (Kpi) in Kp3380. Analysis of a large K. pneumoniae population from 32 European countries showed that the Kpi system is associated with the ST-15 clone. Phylogenetic analysis of the operon revealed that Kpi belongs to the littlecharacterized γ2-fimbrial clade. We demonstrate that Kpi contributes positively to the ability of K.pneumoniae to form biofilms and adhere to different host tissues. Moreover, the in vivo intestinal colonizing capacity of the Kpi-defective mutant was significantly reduced, as was its ability to infect Galleria mellonella. The findings provide information about the pathobiology and epidemicity of Kpi+ K. pneumoniae and indicate that the presence of Kpi may explain the success of the ST-15 clone. Disrupting bacterial adherence to the intestinal surface could potentially target gastrointestinal colonization., This research was supported by Projects p-01216A and IJCI-2016-29524 (to A.P.), funded by the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) and the Minestry of Economy and Competetiveness(MINECO), respectively. It was also supported by Projects PI11/01034 (to M.P.), PI14/00059 and PI17/1482 (to M.P. and A.B.), and PI18/00501 (to G.B.),included in the National Plan for Scientific Research, Development and Technological Innovation 2013-2016 and funded by the Instituto de Salud Carlos III (ISCIII) and Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/006) cofinanced by European Development Regional Fund “A way to achieve Europe” and operative program Intelligent Growth 2014-2020. Grant BFU2016-77835-R of the MINECO (to A.R.) also supported this research. E.G. was financially supported by the SEIMC project. J.C.V.-U. was financially supported by the PFIS (Contratos Predoctorales de Formación en Investigación en Salud) program (F18/00315);J.A.V. was financially supported by IN607A 2016/22; M.M.-G. was financially supported by a Clara Roy grant (SEIMC); A.B. was financially supported by the Miguel Servet program (ISCIII, Spain); B.K.R.-J. was financially supported by Marie S. Curie Action SaPhaDe project (MSCA-IF-GF-836754); and A.P. was financially supported by the Juan de la Cierva program (MINECO, IJCI-2016-29524).

Bacterial genes are typically grouped into operons defined as clusters of adjacent genes encoding for proteins that fill related roles and are transcribed into a single polycistronic mRNA molecule. This simple organization provides an efficient mechanism to coordinate the expression of neighboring genes and is at the basis of gene regulation in bacteria. Here, we report the existence of a higher level of organization in operon structure that we named noncontiguous operon and consists in an operon containing a gene(s) that is transcribed in the opposite direction to the rest of the operon. This transcriptional architecture is exemplified by the genes menE-menC-MW1733-ytkD-MW1731 involved in menaquinone synthesis in the major human pathogen Staphylococcus aureus. We show that menE-menC-ytkD-MW1731 genes are transcribed as a single transcription unit, whereas the MW1733 gene, located between menC and ytkD, is transcribed in the opposite direction. This genomic organization generates overlapping transcripts whose expression is mutually regulated by transcriptional interference and RNase III processing at the overlapping region. In light of our results, the canonical view of operon structure should be revisited by including this operon arrangement in which cotranscription and overlapping transcription are combined to coordinate functionally related gene expression., This work was supported by the Spanish Ministry of Economy and Competitiveness Grants BIO2014-53530-R and BIO2017-83035-R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union). A.T.-A. is supported by the European Research Council under the European Union’s Horizon 2020 research and innovation programme Grant Agreement 646869.

Significance Bacterial pathogens cause plant diseases that threaten the global food supply. To control diseases, it is important to understand how pathogenic bacteria evade plant defense and promote infection. We identify from the phytopathogen Pseudomonas syringae a small-molecule virulence factor—phevamine A. Both the chemical structure and mode of action of phevamine A are different from known bacterial phytotoxins. Phevamine A promotes bacterial growth by suppressing plant immune responses, including both early (the generation of reactive oxygen species) and late (the deposition of cell wall reinforcing callose in leaves and leaf cell death) markers. This work uncovers a widely distributed, small-molecule virulence factor and shows the power of a multidisciplinary approach to identify small molecules important for plant infection., Bacterial plant pathogens cause significant crop damage worldwide. They invade plant cells by producing a variety of virulence factors, including small-molecule toxins and phytohormone mimics. Virulence of the model pathogen Pseudomonas syringae pv. tomato DC3000 (Pto) is regulated in part by the sigma factor HrpL. Our study of the HrpL regulon identified an uncharacterized, three-gene operon in Pto that is controlled by HrpL and related to the Erwinia hrp-associated systemic virulence (hsv) operon. Here, we demonstrate that the hsv operon contributes to the virulence of Pto on Arabidopsis thaliana and suppresses bacteria-induced immune responses. We show that the hsv-encoded enzymes in Pto synthesize a small molecule, phevamine A. This molecule consists of l-phenylalanine, l-valine, and a modified spermidine, and is different from known small molecules produced by phytopathogens. We show that phevamine A suppresses a potentiation effect of spermidine and l-arginine on the reactive oxygen species burst generated upon recognition of bacterial flagellin. The hsv operon is found in the genomes of divergent bacterial genera, including ∼37% of P. syringae genomes, suggesting that phevamine A is a widely distributed virulence factor in phytopathogens. Our work identifies a small-molecule virulence factor and reveals a mechanism by which bacterial pathogens overcome plant defense. This work highlights the power of omics approaches in identifying important small molecules in bacteria–host interactions.

The ferric-uptake regulator (Fur) is an Fe2+-responsive transcription factor that coordinates iron homeostasis in many bacteria. Recently, we reported that expression of the Escherichia coli Fur regulon is also impacted by O2 tension. Here, we show that for most of the Fur regulon, Fur binding and transcriptional repression increase under anaerobic conditions, suggesting that Fur is controlled by O2 availability. We found that the intracellular, labile Fe2+ pool was higher under anaerobic conditions compared with aerobic conditions, suggesting that higher Fe2+ availability drove the formation of more Fe2+-Fur and, accordingly, more DNA binding. O2 regulation of Fur activity required the anaerobically induced FeoABC Fe2+ uptake system, linking increased Fur activity to ferrous import under iron-sufficient conditions. The increased activity of Fur under anaerobic conditions led to a decrease in expression of ferric import systems. However, the combined positive regulation of the feoABC operon by ArcA and FNR partially antagonized Fur-mediated repression of feoABC under anaerobic conditions, allowing ferrous transport to increase even though Fur is more active. This design feature promotes a switch from ferric import to the more physiological relevant ferrous iron under anaerobic conditions. Taken together, we propose that the influence of O2 availability on the levels of active Fur adds a previously undescribed layer of regulation in maintaining cellular iron homeostasis.

While we have come to appreciate the architectural complexity of microbially synthesized secondary metabolites, far less attention has been paid to linking their structural features with possible modes of action. This is certainly the case with tropodithietic acid (TDA), a broad-spectrum antibiotic generated by marine bacteria that engage in dynamic symbioses with microscopic algae. TDA promotes algal health by killing unwanted marine pathogens; however, its mode of action (MoA) and significance for the survival of an algal–bacterial miniecosystem remains unknown. Using cytological profiling, we herein determine the MoA of TDA and surprisingly find that it acts by a mechanism similar to polyether antibiotics, which are structurally highly divergent. We show that like polyether drugs, TDA collapses the proton motive force by a proton antiport mechanism, in which extracellular protons are exchanged for cytoplasmic cations. The α-carboxy-tropone substructure is ideal for this purpose as the proton can be carried on the carboxyl group, whereas the basicity of the tropylium ion facilitates cation export. Based on similarities to polyether anticancer agents we have further examined TDA’s cytotoxicity and find it to exhibit potent, broad-spectrum anticancer activities. These results highlight the power of MoA-profiling technologies in repurposing old drugs for new targets. In addition, we identify an operon that confers TDA resistance to the producing marine bacteria. Bioinformatic and biochemical analyses of these genes lead to a previously unknown metabolic link between TDA/acid resistance and the γ-glutamyl cycle. The implications of this resistance mechanism in the context of the algal-bacterial symbiosis are discussed.

In a number of bacterial pathogens, the production of virulence factors is induced at 37 °C; this effect is often regulated by mRNA structures formed in the 5′ untranslated region (UTR) that block translation initiation of genes at environmental temperatures. At 37 °C, the RNA structures become unstable and ribosomes gain access to their binding sites in the mRNAs. Pseudomonas aeruginosa is an important opportunistic pathogen and the expression of many of its virulence-associated traits is regulated by the quorum-sensing (QS) response, but the effect of temperature on virulence-factor expression is not well-understood. The aim of this work is the characterization of the molecular mechanism involved in thermoregulation of QS-dependent virulence-factor production. We demonstrate that traits that are dependent on the QS transcriptional regulator RhlR have a higher expression at 37 °C, correlating with a higher RhlR concentration as measured by Western blot. We also determined, using gene fusions and point mutations, that RhlR thermoregulation is a posttranscriptional effect dependent on an RNA thermometer of the ROSE (Repression Of heat-Shock gene Expression) family. This RNA element regulates the expression of the rhlAB operon, involved in rhamnolipid production, and of the downstream rhlR gene. We also identified a second functional thermometer in the 5′ UTR of the lasI gene. We confirmed that these RNA thermometers are the main mechanism of thermoregulation of QS-dependent gene expression in P. aeruginosa using quantitative real-time PCR. This is the first description, to our knowledge, of a ROSE element regulating the expression of virulence traits and of an RNA thermometer controlling multiple genes in an operon through a polar effect.

The engineering of complex metabolic pathways requires the concerted expression of multiple genes. In plastids (chloroplasts) of plant cells, genes are organized in operons that are coexpressed as polycistronic transcripts and then often are processed further into monocistronic mRNAs. Here we have used the tocochromanol pathway (providing tocopherols and tocotrienols, collectively also referred to as “vitamin E”) as an example to establish principles of successful multigene engineering by stable transformation of the chloroplast genome, a technology not afflicted with epigenetic variation and/or instability of transgene expression. Testing a series of single-gene constructs (encoding homogentisate phytyltransferase, tocopherol cyclase, and γ-tocopherol methyltransferase) and rationally designed synthetic operons in tobacco and tomato plants, we ( i ) confirmed previous results suggesting homogentisate phytyltransferase as the limiting enzymatic step in the pathway, ( ii ) comparatively characterized the bottlenecks in tocopherol biosynthesis in transplastomic leaves and tomato fruits, and ( iii ) achieved an up to tenfold increase in total tocochromanol accumulation. In addition, our results uncovered an unexpected light-dependent regulatory link between tocochromanol metabolism and the pathways of photosynthetic pigment biosynthesis. The synthetic operon design developed here will facilitate future synthetic biology applications in plastids, especially the design of artificial operons that introduce novel biochemical pathways into plants.

Evolutionary biologists have postulated that several fitness advantages may be conferred by the maintenance of duplicate genes, including environmental adaptation resulting from differential regulation. We examined the expression and physiological contributions of two redundant operons in the adaptable bacterium Pseudomonas aeruginosa PA14. These operons, phzA1-G1 ( phz1 ) and phzA2-G2 ( phz2 ), encode nearly identical sets of proteins that catalyze the synthesis of phenazine-1-carboxylic acid, the precursor for several phenazine derivatives. Phenazines perform diverse roles in P. aeruginosa physiology and act as virulence factors during opportunistic infections of plant and animal hosts. Although reports have indicated that phz1 is regulated by the Pseudomonas quinolone signal, factors controlling phz2 expression have not been identified, and the relative contributions of these redundant operons to phenazine biosynthesis have not been evaluated. We found that in liquid cultures, phz1 was expressed at higher levels than phz2 , although phz2 showed a greater contribution to phenazine production. In colony biofilms, phz2 was expressed at high levels, whereas phz1 expression was not detectable, and phz2 was responsible for virtually all phenazine production. Analysis of mutants defective in quinolone signal synthesis revealed a critical role for 4-hydroxy-2-heptylquinoline in phz2 induction. Finally, deletion of phz2 , but not of phz1 , decreased lung colonization in a murine model of infection. These results suggest that differential regulation of the redundant phz operons allows P. aeruginosa to adapt to diverse environments.

The rapid advance in genome sequencing presents substantial challenges for protein functional assignment, with half or more of new protein sequences inferred from these genomes having uncertain assignments. The assignment of enzyme function in functionally diverse superfamilies represents a particular challenge, which we address through a combination of computational predictions, enzymology, and structural biology. Here we describe the results of a focused investigation of a group of enzymes in the enolase superfamily that are involved in epimerizing dipeptides. The first members of this group to be functionally characterized were Ala-Glu epimerases in Eschericiha coli and Bacillus subtilis , based on the operon context and enzymological studies; these enzymes are presumed to be involved in peptidoglycan recycling. We have subsequently studied more than 65 related enzymes by computational methods, including homology modeling and metabolite docking, which suggested that many would have divergent specificities;, i.e., they are likely to have different (unknown) biological roles. In addition to the Ala-Phe epimerase specificity reported previously, we describe the prediction and experimental verification of: ( i ) a new group of presumed Ala-Glu epimerases; ( ii ) several enzymes with specificity for hydrophobic dipeptides, including one from Cytophaga hutchinsonii that epimerizes D-Ala-D-Ala; and ( iii ) a small group of enzymes that epimerize cationic dipeptides. Crystal structures for certain of these enzymes further elucidate the structural basis of the specificities. The results highlight the potential of computational methods to guide experimental characterization of enzymes in an automated, large-scale fashion.

We show here that the promoters for many of the Escherichia coli ribosomal protein operons are regulated directly by two transcription factors, the small RNA polymerase-binding protein DksA and the nutritional stress-induced nucleotide ppGpp. ppGpp and DksA work together to inhibit transcription initiation from ribosomal protein promoters in vitro and in vivo. The degree of promoter regulation by ppGpp/DksA varies among the r-protein promoters, but some are inhibited almost as much as rRNA promoters. Thus, many r-protein operons are regulated at the level of transcription in addition to their control by the classic translational feedback systems discovered ~30 y ago. We conclude that direct control of r-protein promoters and rRNA promoters by the same signal, ppGpp/DksA, makes a major contribution to the balanced and coordinated synthesis rates of all of the ribosomal components.

Molecular data on a limited number of chromosomal loci have shown that the population of Neisseria meningitidis ( Nm ), a deadly human pathogen, is structured in distinct lineages. Given that the Nm population undergoes substantial recombination, the mechanisms resulting in the evolution of these lineages, their persistence in time, and the implications for the pathogenicity of the bacterium are not yet completely understood. Based on whole-genome sequencing, we show that Nm is structured in phylogenetic clades. Through acquisition of specific genes and through insertions and rearrangements, each clade has acquired and remodeled specific genomic tracts, with the potential to impact on the commensal and virulence behavior of Nm . Despite this clear evidence of a structured population, we confirm high rates of detectable recombination throughout the whole Nm chromosome. However, gene conversion events were found to be longer within clades than between clades, suggesting a DNA cleavage mechanism associated with the phylogeny of the species. We identify 22 restriction modification systems, probably acquired by horizontal gene transfer from outside of the species/genus, whose distribution in the different strains coincides with the phylogenetic clade structure. We provide evidence that these clade-associated restriction modification systems generate a differential barrier to DNA exchange consistent with the observed population structure. These findings have general implications for the emergence of lineage structure and virulence in recombining bacterial populations, and they could provide an evolutionary framework for the population biology of a number of other bacterial species that show contradictory population structure and dynamics.

Chaperonins (CPN) are ubiquitous oligomeric protein machines that mediate the ATP-dependent folding of polypeptide chains. These chaperones have not only been assigned stress response and normal housekeeping functions but also have a role in certain human disease states. A longstanding convention divides CPNs into two groups that share many conserved sequence motifs but differ in both structure and distribution. Group I complexes are the well known GroEL/ES heat-shock proteins in bacteria, that also occur in some species of mesophilic archaea and in the endosymbiotic organelles of eukaryotes. Group II CPNs are found only in the cytosol of archaea and eukaryotes. Here we report a third, divergent group of CPNs found in several species of bacteria. We propose to name these Group III CPNs because of their distant relatedness to both Group I and II CPNs as well as their unique genomic context, within the hsp70 operon. The prototype Group III CPN, Carboxydothermus hydrogenoformans chaperonin ( Ch -CPN), is able to refold denatured proteins in an ATP-dependent manner and is structurally similar to the Group II CPNs, forming a 16-mer with each subunit contributing to a flexible lid domain. The Group III CPN represent a divergent group of bacterial CPNs distinct from the GroEL/ES CPN found in all bacteria. The Group III lineage may represent an ancient horizontal gene transfer from an archaeon into an early Firmicute lineage. An analysis of their functional and structural characteristics may provide important insights into the early history of this ubiquitous family of proteins.

The genus Mycobacterium , which is a member of the high G+C group of Gram-positive bacteria, includes important pathogens, such as M. tuberculosis and M. leprae . A recent publication in PNAS reported that M. marinum and M. bovis bacillus Calmette–Guérin produce a type of spore known as an endospore, which had been observed only in the low G+C group of Gram-positive bacteria. Evidence was presented that the spores were similar to endospores in ultrastructure, in heat resistance and in the presence of dipicolinic acid. Here, we report that the genomes of Mycobacterium species and those of other high G+C Gram-positive bacteria lack orthologs of many, if not all, highly conserved genes diagnostic of endospore formation in the genomes of low G+C Gram-positive bacteria. We also failed to detect the presence of endospores by light microscopy or by testing for heat-resistant colony-forming units in aged cultures of M. marinum . Finally, we failed to recover heat-resistant colony-forming units from frogs chronically infected with M. marinum . We conclude that it is unlikely that Mycobacterium is capable of endospore formation.

d -Amino acids exist in living organisms as specialized components of many different machineries. Biosynthesis of d -amino acids from racemization of predominant l -enantiomers is catalyzed by a single enzyme. Here, we report the finding of a novel 2-component amino acid racemase for d -to- l inversion in d -arginine metabolism of Pseudomonas aeruginosa . From DNA microarray analysis, the putative dauBAR operon (for d -arginine utilization) of unknown functions was found to be highly induced by d -arginine. The importance of the dau operon in d -arginine metabolism was demonstrated by the findings that strains with a lesion at dauA or dauB failed to use d -arginine as sole carbon source. Two lines of evidence suggest that DauA and DauB are required for d -to- l racemization of arginine. First, growth complementation of an l -arginine auxotroph by d -arginine was abolished by a lesion at dauA or dauB . Second, d -arginine induced l -arginine-specific genes in the parental strain PAO1 but not in its dauA or dauB mutants. This hypothesis was further supported by activity measurements of the purified enzymes: DauA catalyzes oxidative deamination of d -arginine into 2-ketoarginine and ammonia, and DauB is able to use 2-ketoarginine and ammonia as substrates and convert them into l -arginine in the presence of NADPH or NADH. Thus, we propose that DauA and DauB are coupled catabolic and anabolic dehydrogenases to perform d -to- l racemization of arginine, which serves as prerequisite of d -arginine utilization through l -arginine catabolic pathways.

Decades ago, the importance of cytokinins (CKs) during Rhodococcus fascians pathology had been acknowledged, and an isopentenyltransferase gene had been characterized in the fas operon of the linear virulence plasmid, but hitherto, no specific CK(s) could be associated with virulence. We show that the CK receptors AHK3 and AHK4 of Arabidopsis thaliana are essential for symptom development, and that the CK perception machinery is induced upon infection, underlining its central role in the symptomatology. Three classical CKs [isopentenyladenine, trans -zeatin, and cis -zeatin (cZ)] and their 2-methylthio (2MeS)-derivatives were identified by CK profiling of both the pathogenic R. fascians strain D188 and its nonpathogenic derivative D188–5. However, the much higher CK levels in strain D188 suggest that the linear plasmid is responsible for the virulence-associated production. All R. fascians CKs were recognized by AHK3 and AHK4, and, although they individually provoked typical CK responses in several bioassays, the mixture of bacterial CKs exhibited clear synergistic effects. The cis - and 2MeS-derivatives were poor substrates of the apoplastic CK oxidase/dehydrogenase enzymes and the latter were not cytotoxic at high concentrations. Consequently, the accumulating 2MeScZ (and cZ) in infected Arabidopsis tissue contribute to the continuous stimulation of tissue proliferation. Based on these results, we postulate that the R. fascians pathology is based on the local and persistent secretion of an array of CKs.

Bacterial operons for F 1 F o -ATP synthase typically include an uncI gene that encodes a function-unknown small hydrophobic protein. When we expressed a hybrid F 1 F o (F 1 from thermophilic Bacillus PS3 and Na + -translocating F o from Propionigenium modestum ) in Escherchia coli cells, we found that uncI derived from P. modestum was indispensable to produce active enzyme; without uncI , c -subunits in F 1 F o existed as monomers but not as functional c 11 -ring. When uncI was expressed from another plasmid at the same time, active F 1 F o with c 11 -ring was produced. A plasmid containing only uncI and c -subunit gene produced c 11 -ring, but a plasmid containing only c -subunit gene did not. Direct interaction of UncI protein with c -subunits was suggested from copurification of His-tagged UncI protein and c -subunits, both in the state of c 11 -ring and c -monomers. Na + induced dissociation of His-tagged UncI protein from c 11 -ring but not from c -monomers. These results show that UncI is a chaperone-like protein that assists c 11 -ring assembly from c -monomers in the membrane.

The transcriptional activator HrpB of the bacterial wilt causing betaproteobacterium Ralstonia solanacearum represents a key regulator for pathogenicity. In particular, it drives expression of hrp genes encoding a type III secretion system (T3SS) as well as effector molecules for delivery into the host cytosol to promote disease. However, the HrpB regulon extends beyond this T3SS. We describe here an HrpB-activated operon of six genes that is responsible for the synthesis of a fluorescent isatin derivative of 149 Amu that we named HDF for HrpB-dependent factor and that we purified from culture supernatants. The structure of the labile molecule was solved by using NMR and CD spectroscopy to be (3 S )-3-hydroxy-indolin-2-one and confirmed by its chemical synthesis and MS spectrometry. HDF was found to be present at 20 nM in wild-type cultures grown on minimal medium, and its synthesis increased 15-fold upon overproduction of HrpB, confirming that HrpB activates HDF synthesis. The addition of tryptophan significantly stimulated HDF biosynthesis and was shown to represent the precursor molecule for HDF synthesis. A search for the biological function of the molecule revealed that HDF induces acyl-homoserine lactone receptor-mediated reporter activity of the well studied LuxR transcriptional regulator of Vibrio fischeri . Thus, our results provide evidence that the specificity of acyl-homoserine lactone (acyl-HSL) receptors is clearly broader than previously considered. The failure to detect induction by HDF of the described endogenous quorum-sensing circuits of the pathogen points to a role in interfering with cell–cell signaling of rivalling bacteria.

A metabolic pathway for biosynthesis of the nonreducing disaccharide mannosylfructose (β-fructofuranosyl-α-mannopyranoside), an important osmolyte in Agrobacterium tumefaciens, was discovered. We have identified and functionally characterized two ORFs that correspond to genes (named mfpsA and mfppA) encoding the rare enzymes mannosylfructose-phosphate synthase and mannosylfructose-phosphate phosphatase, an associated phosphohydrolase. The mfpsA and mfppA genes are arranged in an operon structure, whose transcription is up-regulated by NaCl, resulting in the accumulation of mannosylfructose in the cells. Not only is the biosynthesis of mannosylfructose mechanistically similar to that of sucrose, but the corresponding genes for the biosynthesis of both disaccharides are also phylogenetic close relatives. Importantly, a protein phylogeny analysis indicated that mannosylfructose-phosphate synthase defines a unique group of mannosyltransferases. © 2007 by The National Academy of Sciences of the USA. Fil: Torres, Leticia Luciana. Fundación para Investigaciones Biológicas Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina Fil: Salerno, Graciela Lidia. Fundación para Investigaciones Biológicas Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina

Transduction frequency with phage P1 had been observed to be very low in Escherichia coli K-12 mutants lacking the operon ( ppk1-ppx ) responsible for the synthesis of inorganic polyphosphate (poly P). We now find that these mutants, for lack of poly P, are lysogenic for P1 and when infected with phage P1 produce only ≈1% the number of infective centers compared with the WT host. Both phage adsorption and release were unaffected. The host-encoded P1 late-gene transcriptional activator, SspA, failed to show the transcriptional increase in the mutant, observed in the WT. UV induction of a P1-infected mutant resulted in a 200-fold increase in the production of infectious phage particles. The lysogenized P1 (P1 mut ) and P1 progeny from the mutant host (Δ ppk1-ppx ) produced plaques of differing morphologies, whereas P1 progeny from the WT yielded only small, clear plaques. Two discernable variants, one producing small and clear plaques (P1 small ) and the other large plaques with turbid rims (P1 large ), had broader host range and produced larger burst sizes in WT compared with P1. Transmission electron microscopy showed P1 mut had contractile sheath defects. Thus, the lack of poly P/PPK1 in the mutant host resulted in the formation of defective P1 particles during intracellular growth. A filamentous phage, fd, also failed to produce plaques on a mutant lawn. Although fd adsorbed to the F-pilus, its DNA failed to enter the mutant host.

Coastal aquatic environments are typically more highly productive and dynamic than open ocean ones. Despite these differences, cyanobacteria from the genus Synechococcus are important primary producers in both types of ecosystems. We have found that the genome of a coastal cyanobacterium, Synechococcus sp. strain CC9311, has significant differences from an open ocean strain, Synechococcus sp. strain WH8102, and these are consistent with the differences between their respective environments. CC9311 has a greater capacity to sense and respond to changes in its (coastal) environment. It has a much larger capacity to transport, store, use, or export metals, especially iron and copper. In contrast, phosphate acquisition seems less important, consistent with the higher concentration of phosphate in coastal environments. CC9311 is predicted to have differences in its outer membrane lipopolysaccharide, and this may be characteristic of the speciation of some cyanobacterial groups. In addition, the types of potentially horizontally transferred genes are markedly different between the coastal and open ocean genomes and suggest a more prominent role for phages in horizontal gene transfer in oligotrophic environments.

The enormous complexity of biological networks has led to the suggestion that networks are built of modules that perform particular functions and are “reused” in evolution in a manner similar to reusable domains in protein structures or modules of electronic circuits. Analysis of known biological networks has revealed several modules, many of which have transparent biological functions. However, it remains to be shown that identified structural modules constitute evolutionary building blocks, independent and easily interchangeable units. An alternative possibility is that evolutionary modules do not match structural modules. To investigate the structure of evolutionary modules and their relationship to functional ones, we integrated a metabolic network with evolutionary associations between genes inferred from comparative genomics. The resulting metabolic–genomic network places metabolic pathways into evolutionary and genomic context, thereby revealing previously unknown components and modules. We analyzed the integrated metabolic–genomic network on three levels: macro-, meso-, and microscale. The macroscale level demonstrates strong associations between neighboring enzymes and between enzymes that are distant on the network but belong to the same linear pathway. At the mesoscale level, we identified evolutionary metabolic modules and compared them with traditional metabolic pathways. Although, in some cases, there is almost exact correspondence, some pathways are split into independent modules. On the microscale level, we observed high association of enzyme subunits and weak association of isoenzymes independently catalyzing the same reaction. This study shows that evolutionary modules, rather than pathways, may be thought of as regulatory and functional units in bacterial genomes.

The b1012 operon of Escherichia coli K-12, which is composed of seven unidentified ORFs, is one of the most highly expressed operons under control of nitrogen regulatory protein C. Examination of strains with lesions in this operon on Biolog Phenotype MicroArray (PM3) plates and subsequent growth tests indicated that they failed to use uridine or uracil as the sole nitrogen source and that the parental strain could use them at room temperature but not at 37°C. A strain carrying an ntrB (Con) mutation, which elevates transcription of genes under nitrogen regulatory protein C control, could also grow on thymidine as the sole nitrogen source, whereas strains with lesions in the b1012 operon could not. Growth-yield experiments indicated that both nitrogens of uridine and thymidine were available. Studies with [ 14 C]uridine indicated that a three-carbon waste product from the pyrimidine ring was excreted. After trimethylsilylation and gas chromatography, the waste product was identified by mass spectrometry as 3-hydroxypropionic acid. In agreement with this finding, 2-methyl-3-hydroxypropionic acid was released from thymidine. Both the number of available nitrogens and the waste products distinguished the pathway encoded by the b1012 operon from pyrimidine catabolic pathways described previously. We propose that the genes of this operon be named rutA–G for py r imidine ut ilization. The product of the divergently transcribed gene, b1013, is a tetracycline repressor family regulator that controls transcription of the b1012 operon negatively.

Teichoic acids (TAs), and especially lipoteichoic acids (LTAs), are one of the main immunostimulatory components of pathogenic Gram-positive bacteria. Their contribution to the immunomodulatory properties of commensal bacteria and especially of lactic acid bacteria has not yet been investigated in detail. To evaluate the role of TAs in the interaction between lactic acid bacteria and the immune system, we analyzed the antiinflammatory properties of a mutant of Lactobacillus plantarum NCIMB8826 affected in the TA biosynthesis pathway both in vitro (mononuclear cells stimulation) and in vivo (murine model of colitis). This Dlt - mutant was found to incorporate much less d -Ala in its TAs than the WT strain. This defect significantly impacted the immunomodulation reactions induced by the bacterium, as shown by a dramatically reduced secretion of proinflammatory cytokines by peripheral blood mononuclear cells and monocytes stimulated by the Dlt - mutant as compared with the parental strain. Concomitantly, a significant increase in IL-10 production was stimulated by the Dlt - mutant in comparison with the WT strain. Moreover, the proinflammatory capacity of L. plantarum -purified LTA was found to be Toll-like receptor 2-dependent. Consistent with the in vitro results, the Dlt - mutant was significantly more protective in a murine colitis model than its WT counterpart. The results indicated that composition of LTA within the whole-cell context of L. plantarum can modulate proinflammatory or antiinflammatory immune responses.

In the methanogenic archaeon Methanosarcina barkeri Fusaro, the N 5 -methyl-tetrahydrosarcinapterin (CH 3 -H 4 SPT):coenzyme M (CoM) methyltransferase, encoded by the mtr operon, catalyzes the energy-conserving (sodium-pumping) methyl transfer from CH 3 -H 4 SPT to CoM during growth on H 2 /CO 2 or acetate. However, in the disproportionation of C-1 compounds, such as methanol, to methane and carbon dioxide, it catalyzes the reverse, endergonic transfer from methyl-CoM to H 4 SPT, which is driven by sodium uptake. It has been proposed that a bypass for this energy-consuming reaction may occur via a direct methyl transfer from methanol to H 4 SPT. To test this, an mtr deletion mutant was constructed and characterized in M. barkeri Fusaro. The mutant is unable to grow on methanol, acetate or H 2 /CO 2 , but can grow on methanol with H 2 /CO 2 and, surprisingly, methanol with acetate. 13 C labeling experiments show that growth on acetate with methanol involves a previously unknown methanogenic pathway, in which oxidation of acetate to a mixture of CO 2 and formic acid is coupled to methanol reduction. Interestingly, although the mutant is unable to grow on methanol alone, it remains capable of producing methane from this substrate. Thus, the proposed Mtr bypass does exist, but is unable to support growth of the organism.

Bacterial-host attachment by means of bacterial adhesins is a key step in host colonization. Phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (mainly on-to-off switching). fimB is separated from the divergent yjhATS operon by a large (1.4 kbp) intergenic region. Short (≈28 bp) cis-active elements (regions 1 and 2) close to yjhA stimulate fimB expression and are required for sialic acid (Neu 5 Ac) sensitivity of its expression [El-Labany, S., Sohanpal, B. K., Lahooti, M., Akerman, R. & Blomfield, I. C. (2003) Mol. Microbiol. 49, 1109-1118]. Here, we show that whereas NanR, a sialic acid-response regulator, binds to region 1, NagC, a GlcNAc-6P-responsive protein, binds to region 2 instead. The NanR- and NagC-binding sites lie adjacent to deoxyadenosine methylase (Dam) methylation sites (5′-GATC) that are protected from modification, and the two regulators are shown to be required for methylation protection at regions 1 and 2, respectively. Mutations in nanR and nagC diminish fimB expression, and both fimB expression and FimB recombination are inhibited by GlcNAc (3- and >35-fold, respectively). Sialic acid catabolism generates GlcNAc-6-P, and whereas GlcNAc disrupts methylation protection by NagC alone, Neu 5 Ac inhibits the protection mediated by both NanR and NagC as expected. Type 1 fimbriae are proinflammatory, and host defenses enhance the release of both Neu 5 Ac and GlcNAc by a variety of mechanisms. Inhibition of type 1 fimbriation by these amino sugars may thus help balance the interaction between E. coli and its hosts.

Chitin, a highly insoluble polymer of GlcNAc, is produced in massive quantities in the marine environment. Fortunately for survival of aquatic ecosystems, chitin is rapidly catabolized by marine bacteria. Here we describe a bacterial two-component hybrid sensor/kinase (of the ArcB type) that rigorously controls expression of ≈50 genes, many involved in chitin degradation. The sensor gene, chi S, was identified in Vibrio furnissii and Vibrio cholerae (predicted amino acid sequences, full-length: 84% identical, 93% similar). Mutants of chi S grew normally on GlcNAc but did not express extracellular chitinase, a specific chitoporin, or β-hexosaminidases, nor did they exhibit chemotaxis, transport, or growth on chitin oligosaccharides such as (GlcNAc) 2 . Expression of these systems requires three components: wild-type chi S; a periplasmic high-affinity c hitin oligosaccharide, (GlcNAc) n ( n > 1), b inding p rotein (CBP); and the environmental signal, (GlcNAc) n . Our data are consistent with the following model. In the uninduced state, CBP binds to the periplasmic domain of ChiS and “locks” it into the minus conformation. The environmental signal, (GlcNAc) n , dissociates the complex by binding to CBP, releasing ChiS, yielding the plus phenotype (expression of chitinolytic genes). In V. cholerae , a cluster of 10 contiguous genes (VC0620–VC0611) apparently comprise a (GlcNAc) 2 catabolic operon. CBP is encoded by the first, VC0620, whereas VC0619–VC0616 encode a (GlcNAc) 2 ABC-type permease. Regulation of chi S requires expression of CBP but not (GlcNAc) 2 transport. (GlcNAc) n is suggested to be essential for signaling these cells that chitin is in the microenvironment.

The rnpA mutation, A49, in Escherichia coli reduces the level of RNase P at 43°C because of a temperature-sensitive mutation in C5 protein, the protein subunit of the enzyme. Microarray analysis reveals the expression of several noncoding intergenic regions that are increased at 43°C compared with 30°C. These regions are substrates for RNase P, and they are cleaved less efficiently than, for example, tRNA precursors. An analysis of the tna, secG, rbs , and his operons, all of which contain RNase P cleavage sites, indicates that RNase P affects gene expression for regions downstream of its cleavage sites.

For more than a decade, it has been recognized that arsenate [H 2 AsO 4 1- ; As(V)] can be used by microorganisms as a terminal electron acceptor in anaerobic respiration. Given the toxicity of arsenic, the mechanistic basis of this process is intriguing, as is its evolutionary origin. Here we show that a two-gene cluster ( arrAB ; a rsenate r espiratory r eduction) in the bacterium Shewanella sp. strain ANA-3 specifically confers respiratory As(V) reductase activity. Mutants with in-frame deletions of either arrA or arrB are incapable of growing on As(V), yet both are able to grow on a wide variety of other electron acceptors as efficiently as the wild-type. Complementation by the wild-type sequence rescues the mutants' ability to respire As(V). arrA is predicted to encode a 95.2-kDa protein with sequence motifs similar to the molybdenum containing enzymes of the dimethyl sulfoxide reductase family. arrB is predicted to encode a 25.7-kDa iron–sulfur protein. arrA and arrB comprise an operon that contains a twin arginine translocation (Tat) motif in ArrA (but not in ArrB) as well as a putative anaerobic transcription factor binding site upstream of arrA , suggesting that the respiratory As(V) reductase is exported to the periplasm via the Tat pathway and under anaerobic transcriptional control. These genes appear to define a new class of reductases that are specific for respiratory As(V) reduction.

Ansamycins such as rifamycin, ansamitocin, and geldanamycin are an important class of polyketide natural products. Their biosynthetic pathways are especially complex because they involve the formation of 3-amino-5-hydroxybenzoic acid (AHBA) followed by backbone assembly by a hybrid nonribosomal peptide synthetase/polyketide synthase. We have reconstituted the ability to synthesize 2,6-dimethyl-3,5,7-trihydroxy-7-(3′-amino-5′-hydroxyphenyl)-2,4-heptadienoic acid (P8/1-OG), an intermediate in rifamycin biosynthesis, in an extensively manipulated strain of Escherichia coli . The parent strain, BAP1, contains the sfp phosphopantetheinyl transferase gene from Bacillus subtilis , which posttranslationally modifies polyketide synthase and nonribosomal peptide synthetase modules. AHBA biosynthesis in this host required introduction of seven genes from Amycolatopsis mediterranei , which produces rifamycin, and Actinosynnema pretiosum , which produces ansamitocin. Because the four-module RifA protein (530 kDa) from the rifamycin synthetase could not be efficiently produced in an intact form in E. coli , it was genetically split into two bimodular proteins separated by matched linker pairs to facilitate efficient inter-polypeptide transfer of a biosynthetic intermediate. A derivative of BAP1 was engineered that harbors the AHBA biosynthetic operon, the bicistronic RifA construct and the pccB and accA1 genes from Streptomyces coelicolor , which enable methylmalonyl-CoA biosynthesis. Fermentation of this strain of E. coli yielded P8/1-OG, an N -acetyl P8/1-OG analog, and AHBA. In addition to providing a fundamentally new route to shikimate and ansamycin-type compounds, this result enables further genetic manipulation of AHBA-derived polyketide natural products with unprecedented power.

The thiCOGE genes of Rhizobium etli code for enzymes involved in thiamin biosynthesis. These genes are transcribed with a 211-base untranslated leader that contains the thi box, a 38-base sequence highly conserved in the 5' regions of thiamin biosynthetic and transport genes of Gram-positive and Gram-negative organisms. A deletion analysis of thiC-lacZ fusions revealed an unexpected relationship between the degree of repression shown by the deleted derivatives and the length of the thiC sequences present in the transcript. Three regions were found to be important for regulation: (i) the thi box sequence, which is absolutely necessary for high-level expression of thiC; (ii) the region immediately upstream to the translation start codon of thiC, which can be folded into a stem-loop structure that would mask the Shine-Dalgarno sequence; and (iii) the proximal part of the coding region of thiC, which was shown to contain a putative Rho-independent terminator. A comparative phylogenetic analysis revealed a possible folding of the thi box sequence into a hairpin structure composed of a hairpin loop, two helices, and an interior loop. Our results show that thiamin regulation of gene expression involves a complex posttranscriptional mechanism and that the thi box RNA structure is indispensable for thiCOGE expression.

DNA microarrays constructed with full length ORFs from Shewanella oneidensis , MR-1, were hybridized with genomic DNA from nine other Shewanella species and Escherichia coli K-12. This approach enabled visualization of relationships between organisms by comparing individual ORF hybridizations to 164 genes and is further amenable to high-density high-throughput analyses of complete microbial genomes. Conserved genes ( arc A and ATP synthase) were identified among all species investigated. The mtr operon, which is involved in iron reduction, was poorly conserved among other known metal-reducing Shewanella species. Results were most informative for closely related organisms with small subunit rRNA sequence similarities greater than 93% and gyr B sequence similarities greater than 80%. At this level of relatedness, the similarity between hybridization profiles was strongly correlated with sequence divergence in the gyr B gene. Results revealed that two strains of S. oneidensis (MR-1 and DLM7) were nearly identical, with only 3% of the ORFs hybridizing poorly, in contrast to hybridizations with Shewanella putrefaciens , formerly considered to be the same species as MR-1, in which 63% of the ORFs hybridized poorly (log ratios below −0.75). Genomic hybridizations showed that genes in operons had consistent levels of hybridization across an operon in comparison to a randomly sampled data set, suggesting that similar applications will be informative for identification of horizontally acquired genes. The full value of microbial genomic hybridizations lies in providing the ability to understand and display specific differences between closely related organisms providing a window into understanding microheterogeneity, bacterial speciation, and taxonomic relationships.

The thiCOGE genes of Rhizobium etli code for enzymes involved in thiamin biosynthesis. These genes are transcribed with a 211-base untranslated leader that contains the thi box, a 38-base sequence highly conserved in the 5′ regions of thiamin biosynthetic and transport genes of Gram-positive and Gram-negative organisms. A deletion analysis of thiC-lacZ fusions revealed an unexpected relationship between the degree of repression shown by the deleted derivatives and the length of the thiC sequences present in the transcript. Three regions were found to be important for regulation: ( i ) the thi box sequence, which is absolutely necessary for high-level expression of thiC ; ( ii ) the region immediately upstream to the translation start codon of thiC , which can be folded into a stem-loop structure that would mask the Shine-Dalgarno sequence; and ( iii ) the proximal part of the coding region of thiC , which was shown to contain a putative Rho-independent terminator. A comparative phylogenetic analysis revealed a possible folding of the thi box sequence into a hairpin structure composed of a hairpin loop, two helixes, and an interior loop. Our results show that thiamin regulation of gene expression involves a complex posttranscriptional mechanism and that the thi box RNA structure is indispensable for thiCOGE expression.