Publications by authors named "Rocío Canals"

35 Publications

Stepwise evolution of Salmonella Typhimurium ST313 causing bloodstream infection in Africa.

Nat Microbiol 2021 Mar 21;6(3):327-338. Epub 2020 Dec 21.

Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, UK.

Bloodstream infections caused by nontyphoidal Salmonella are a major public health concern in Africa, causing ~49,600 deaths every year. The most common Salmonella enterica pathovariant associated with invasive nontyphoidal Salmonella disease is Salmonella Typhimurium sequence type (ST)313. It has been proposed that antimicrobial resistance and genome degradation has contributed to the success of ST313 lineages in Africa, but the evolutionary trajectory of such changes was unclear. Here, to define the evolutionary dynamics of ST313, we sub-sampled from two comprehensive collections of Salmonella isolates from African patients with bloodstream infections, spanning 1966 to 2018. The resulting 680 genome sequences led to the discovery of a pan-susceptible ST313 lineage (ST313 L3), which emerged in Malawi in 2016 and is closely related to ST313 variants that cause gastrointestinal disease in the United Kingdom and Brazil. Genomic analysis revealed degradation events in important virulence genes in ST313 L3, which had not occurred in other ST313 lineages. Despite arising only recently in the clinic, ST313 L3 is a phylogenetic intermediate between ST313 L1 and L2, with a characteristic accessory genome. Our in-depth genotypic and phenotypic characterization identifies the crucial loss-of-function genetic events that occurred during the stepwise evolution of invasive S. Typhimurium across Africa.
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http://dx.doi.org/10.1038/s41564-020-00836-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018540PMC
March 2021

Genetic variation in the MacAB-TolC efflux pump influences pathogenesis of invasive Salmonella isolates from Africa.

PLoS Pathog 2020 08 24;16(8):e1008763. Epub 2020 Aug 24.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

The various sub-species of Salmonella enterica cause a range of disease in human hosts. The human-adapted Salmonella enterica serovar Typhi enters the gastrointestinal tract and invades systemic sites to cause enteric (typhoid) fever. In contrast, most non-typhoidal serovars of Salmonella are primarily restricted to gut tissues. Across Africa, invasive non-typhoidal Salmonella (iNTS) have emerged with an ability to spread beyond the gastrointestinal tract and cause systemic bloodstream infections with increased morbidity and mortality. To investigate this evolution in pathogenesis, we compared the genomes of African iNTS isolates with other Salmonella enterica serovar Typhimurium and identified several macA and macB gene variants unique to African iNTS. MacAB forms a tripartite efflux pump with TolC and is implicated in Salmonella pathogenesis. We show that macAB transcription is upregulated during macrophage infection and after antimicrobial peptide exposure, with macAB transcription being supported by the PhoP/Q two-component system. Constitutive expression of macAB improves survival of Salmonella in the presence of the antimicrobial peptide C18G. Furthermore, these macAB variants affect replication in macrophages and influence fitness during colonization of the murine gastrointestinal tract. Importantly, the infection outcome resulting from these macAB variants depends upon both the Salmonella Typhimurium genetic background and the host gene Nramp1, an important determinant of innate resistance to intracellular bacterial infection. The variations we have identified in the MacAB-TolC efflux pump in African iNTS may reflect evolution within human host populations that are compromised in their ability to clear intracellular Salmonella infections.
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http://dx.doi.org/10.1371/journal.ppat.1008763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446830PMC
August 2020

Evasion of MAIT cell recognition by the African Typhimurium ST313 pathovar that causes invasive disease.

Proc Natl Acad Sci U S A 2020 08 11;117(34):20717-20728. Epub 2020 Aug 11.

Medical Research Council Human Immunology Unit, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS Oxford, United Kingdom.

Mucosal-associated invariant T (MAIT) cells are innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defense. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with selective absence of MAIT cells have not been identified. We report that typhoidal and nontyphoidal strains activate MAIT cells. However, Typhimurium sequence type 313 (ST313) lineage 2 strains, which are responsible for the burden of multidrug-resistant nontyphoidal invasive disease in Africa, escape MAIT cell recognition through overexpression of This bacterial gene encodes the 4-dihydroxy-2-butanone-4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. The MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of Typhimurium ST313 lineage 2.
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http://dx.doi.org/10.1073/pnas.2007472117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456131PMC
August 2020

A window into lysogeny: revealing temperate phage biology with transcriptomics.

Microb Genom 2020 02 5;6(2). Epub 2020 Feb 5.

Institute of Integrative Biology, University of Liverpool, Liverpool, UK.

Prophages are integrated phage elements that are a pervasive feature of bacterial genomes. The fitness of bacteria is enhanced by prophages that confer beneficial functions such as virulence, stress tolerance or phage resistance, and these functions are encoded by 'accessory' or 'moron' loci. Whilst the majority of phage-encoded genes are repressed during lysogeny, accessory loci are often highly expressed. However, it is challenging to identify novel prophage accessory loci from DNA sequence data alone. Here, we use bacterial RNA-seq data to examine the transcriptional landscapes of five prophages. We show that transcriptomic data can be used to heuristically enrich for prophage features that are highly expressed within bacterial cells and represent functionally important accessory loci. Using this approach, we identify a novel antisense RNA species in prophage BTP1, STnc6030, which mediates superinfection exclusion of phage BTP1. Bacterial transcriptomic datasets are a powerful tool to explore the molecular biology of temperate phages.
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http://dx.doi.org/10.1099/mgen.0.000330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067206PMC
February 2020

The fitness landscape of the African Salmonella Typhimurium ST313 strain D23580 reveals unique properties of the pBT1 plasmid.

PLoS Pathog 2019 09 27;15(9):e1007948. Epub 2019 Sep 27.

Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.

We have used a transposon insertion sequencing (TIS) approach to establish the fitness landscape of the African Salmonella enterica serovar Typhimurium ST313 strain D23580, to complement our previous comparative genomic and functional transcriptomic studies. We used a genome-wide transposon library with insertions every 10 nucleotides to identify genes required for survival and growth in vitro and during infection of murine macrophages. The analysis revealed genomic regions important for fitness under two in vitro growth conditions. Overall, 724 coding genes were required for optimal growth in LB medium, and 851 coding genes were required for growth in SPI-2-inducing minimal medium. These findings were consistent with the essentiality analyses of other S. Typhimurium ST19 and S. Typhi strains. The global mutagenesis approach also identified 60 sRNAs and 413 intergenic regions required for growth in at least one in vitro growth condition. By infecting murine macrophages with the transposon library, we identified 68 genes that were required for intra-macrophage replication but did not impact fitness in vitro. None of these genes were unique to S. Typhimurium D23580, consistent with a high conservation of gene function between S. Typhimurium ST313 and ST19 and suggesting that novel virulence factors are not involved in the interaction of strain D23580 with murine macrophages. We discovered that transposon insertions rarely occurred in many pBT1 plasmid-encoded genes (36), compared with genes carried by the pSLT-BT virulence plasmid and other bacterial plasmids. The key essential protein encoded by pBT1 is a cysteinyl-tRNA synthetase, and our enzymological analysis revealed that the plasmid-encoded CysRSpBT1 had a lower ability to charge tRNA than the chromosomally-encoded CysRSchr enzyme. The presence of aminoacyl-tRNA synthetases in plasmids from a range of Gram-negative and Gram-positive bacteria suggests that plasmid-encoded essential genes are more common than had been appreciated.
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http://dx.doi.org/10.1371/journal.ppat.1007948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785131PMC
September 2019

The use of chicken and insect infection models to assess the virulence of African Salmonella Typhimurium ST313.

PLoS Negl Trop Dis 2019 07 26;13(7):e0007540. Epub 2019 Jul 26.

Department of Functional and Comparative Genomics, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.

Over recent decades, Salmonella infection research has predominantly relied on murine infection models. However, in many cases the infection phenotypes of Salmonella pathovars in mice do not recapitulate human disease. For example, Salmonella Typhimurium ST313 is associated with enhanced invasive infection of immunocompromised people in Africa, but infection of mice and other animal models with ST313 have not consistently reproduced this invasive phenotype. The introduction of alternative infection models could help to improve the quality and reproducibility of pathogenesis research by facilitating larger-scale experiments. To investigate the virulence of S. Typhimurium ST313 in comparison with ST19, a combination of avian and insect disease models were used. We performed experimental infections in five lines of inbred and one line of outbred chickens, as well as in the alternative chick embryo and Galleria mellonella wax moth larvae models. This extensive set of experiments identified broadly similar patterns of disease caused by the African and global pathovariants of Salmonella Typhimurium in the chicken, the chicken embryo and insect models. A comprehensive analysis of all the chicken infection experiments revealed that the African ST313 isolate D23580 had a subtle phenotype of reduced levels of organ colonisation in inbred chickens, relative to ST19 strain 4/74. ST313 isolate D23580 also caused reduced mortality in chicken embryos and insect larvae, when compared with ST19 4/74. We conclude that these three infection models do not reproduce the characteristics of the systemic disease caused by S. Typhimurium ST313 in humans.
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http://dx.doi.org/10.1371/journal.pntd.0007540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685681PMC
July 2019

The diversity, evolution and ecology of Salmonella in venomous snakes.

PLoS Negl Trop Dis 2019 06 4;13(6):e0007169. Epub 2019 Jun 4.

Department of Functional and Comparative Genomics, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.

Background: Reptile-associated Salmonella bacteria are a major, but often neglected cause of both gastrointestinal and bloodstream infection in humans globally. The diversity of Salmonella enterica has not yet been determined in venomous snakes, however other ectothermic animals have been reported to carry a broad range of Salmonella bacteria. We investigated the prevalence and diversity of Salmonella in a collection of venomous snakes and non-venomous reptiles.

Methodology/principle Findings: We used a combination of selective enrichment techniques to establish a unique dataset of reptilian isolates to study Salmonella enterica species-level evolution and ecology and used whole-genome sequencing to investigate the relatedness of phylogenetic groups. We observed that 91% of venomous snakes carried Salmonella, and found that a diverse range of serovars (n = 58) were carried by reptiles. The Salmonella serovars belonged to four of the six Salmonella enterica subspecies: diarizonae, enterica, houtanae and salamae. Subspecies enterica isolates were distributed among two distinct phylogenetic clusters, previously described as clade A (52%) and clade B (48%). We identified metabolic differences between S. diarizonae, S. enterica clade A and clade B involving growth on lactose, tartaric acid, dulcitol, myo-inositol and allantoin.

Significance: We present the first whole genome-based comparative study of the Salmonella bacteria that colonise venomous and non-venomous reptiles and shed new light on Salmonella evolution. Venomous snakes examined in this study carried a broad range of Salmonella, including serovars which have been associated with disease in humans such as S. Enteritidis. The findings raise the possibility that venomous snakes could be a reservoir for Salmonella serovars associated with human salmonellosis.
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http://dx.doi.org/10.1371/journal.pntd.0007169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548357PMC
June 2019

Adding function to the genome of African Salmonella Typhimurium ST313 strain D23580.

PLoS Biol 2019 01 15;17(1):e3000059. Epub 2019 Jan 15.

Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.

Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.
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http://dx.doi.org/10.1371/journal.pbio.3000059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333337PMC
January 2019

Role of a single noncoding nucleotide in the evolution of an epidemic African clade of .

Proc Natl Acad Sci U S A 2018 03 27;115(11):E2614-E2623. Epub 2018 Feb 27.

Institute of Integrative Biology, University of Liverpool, L69 7ZB Liverpool, United Kingdom;

serovar Typhimurium ST313 is a relatively newly emerged sequence type that is causing a devastating epidemic of bloodstream infections across sub-Saharan Africa. Analysis of hundreds of genomes has revealed that ST313 is closely related to the ST19 group of Typhimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by only ∼1,000 SNPs. We hypothesized that the phenotypic differences that distinguish African from ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes. Here we identified 3,597 transcriptional start sites of the ST313 strain D23580, and searched for a gene-expression signature linked to pathogenesis of We identified a SNP in the promoter of the gene that caused high expression of the PgtE virulence factor in African Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance, and modulated virulence in the chicken infection model. We propose that high levels of PgtE expression by African Typhimurium ST313 promote bacterial survival and dissemination during human infection. Our finding of a functional role for an extragenic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on noncoding regions of the genome.
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http://dx.doi.org/10.1073/pnas.1714718115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856525PMC
March 2018

Characterization of the Prophage Repertoire of African Typhimurium ST313 Reveals High Levels of Spontaneous Induction of Novel Phage BTP1.

Front Microbiol 2017 23;8:235. Epub 2017 Feb 23.

Institute of Integrative Biology, University of Liverpool Liverpool, UK.

In the past 30 years, bloodstream infections have become a significant health problem in sub-Saharan Africa and are responsible for the deaths of an estimated 390,000 people each year. The disease is predominantly caused by a recently described sequence type of Typhimurium: ST313, which has a distinctive set of prophage sequences. We have thoroughly characterized the ST313-associated prophages both genetically and experimentally. ST313 representative strain D23580 contains five full-length prophages: BTP1, Gifsy-2, ST64B, Gifsy-1, and BTP5. We show that common Typhimurium prophages Gifsy-2, Gifsy-1, and ST64B are inactivated in ST313 by mutations. Prophage BTP1 was found to be a functional novel phage, and the first isolate of the proposed new species "Salmonella virus BTP1", belonging to the genus. Surprisingly, ∼10 BTP1 virus particles ml were detected in the supernatant of non-induced, stationary-phase cultures of strain D23580, representing the highest spontaneously induced phage titer so far reported for a bacterial prophage. High spontaneous induction is shown to be an intrinsic property of prophage BTP1, and indicates the phage-mediated lysis of around 0.2% of the lysogenic population. The fact that BTP1 is highly conserved in ST313 poses interesting questions about the potential fitness costs and benefits of novel prophages in epidemic Typhimurium ST313.
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http://dx.doi.org/10.3389/fmicb.2017.00235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322425PMC
February 2017

Salmonella Persistence in Tomatoes Requires a Distinct Set of Metabolic Functions Identified by Transposon Insertion Sequencing.

Appl Environ Microbiol 2017 03 15;83(5). Epub 2017 Feb 15.

Soil and Water Science Department, Genetics Institute, University of Florida-IFAS, Gainesville, Florida, USA.

Human enteric pathogens, such as spp. and verotoxigenic , are increasingly recognized as causes of gastroenteritis outbreaks associated with the consumption of fruits and vegetables. Persistence in plants represents an important part of the life cycle of these pathogens. The identification of the full complement of genes involved in the colonization of the model plant (tomato) was carried out using transposon insertion sequencing analysis. With this approach, 230,000 transposon insertions were screened in tomato pericarps to identify loci with reduction in fitness, followed by validation of the screen results using competition assays of the isogenic mutants against the wild type. A comparison with studies in animals revealed a distinct plant-associated set of genes, which only partially overlaps with the genes required to elicit disease in animals. biosynthesis of amino acids was critical to persistence within tomatoes, while amino acid scavenging was prevalent in animal infections. Fitness reduction of the amino acid synthesis mutants was generally more severe in the tomato mutant, which hyperaccumulates certain amino acids, suggesting that these nutrients remain unavailable to spp. within plants. lipopolysaccharide (LPS) was required for persistence in both animals and plants, exemplifying some shared pathogenesis-related mechanisms in animal and plant hosts. Similarly to phytopathogens, spp. required biosynthesis of amino acids, LPS, and nucleotides to colonize tomatoes. Overall, however, it appears that while shares some strategies with phytopathogens and taps into its animal virulence-related functions, colonization of tomatoes represents a distinct strategy, highlighting this pathogen's flexible metabolism. Outbreaks of gastroenteritis caused by human pathogens have been increasingly associated with foods of plant origin, with tomatoes being one of the common culprits. Recent studies also suggest that these human pathogens can use plants as alternate hosts as a part of their life cycle. While dual (animal/plant) lifestyles of other members of the family are well known, the strategies with which colonizes plants are only partially understood. Therefore, we undertook a high-throughput characterization of the functions required for persistence within tomatoes. The results of this study were compared with what is known about genes required for virulence in animals and interactions of plant pathogens with their hosts to determine whether repurposes its virulence repertoire inside plants or whether it behaves more as a phytopathogen during plant colonization. Even though utilized some of its virulence-related genes in tomatoes, plant colonization required a distinct set of functions.
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http://dx.doi.org/10.1128/AEM.03028-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311394PMC
March 2017

Hha has a defined regulatory role that is not dependent upon H-NS or StpA.

Front Microbiol 2015 30;6:773. Epub 2015 Jul 30.

Departament de Microbiologia, Universitat de Barcelona Barcelona, Spain.

The Hha family of proteins is involved in the regulation of gene expression in enterobacteria by forming complexes with H-NS-like proteins. Whereas several amino acid residues of both proteins participate in the interaction, some of them play a key role. Residue D48 of Hha protein is essential for the interaction with H-NS, thus the D48N substitution in Hha protein abrogates H-NS/Hha interaction. Despite being a paralog of H-NS protein, StpA interacts with HhaD48N with higher affinity than with the wild type Hha protein. To analyze whether Hha is capable of acting independently of H-NS and StpA, we conducted transcriptomic analysis on the hha and stpA deletion strains and the hhaD48N substitution strain of Salmonella Typhimurium using a custom microarray. The results obtained allowed the identification of 120 genes regulated by Hha in an H-NS/StpA-independent manner, 38% of which are horizontally acquired genes. A significant number of the identified genes are involved in functions related to cell motility, iron uptake, and pathogenicity. Thus, motility assays, siderophore detection and intra-macrophage replication assays were performed to confirm the transcriptomic data. Our findings point out the importance of Hha protein as an independent regulator in S. Typhimurium, highlighting a regulatory role on virulence.
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http://dx.doi.org/10.3389/fmicb.2015.00773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519777PMC
August 2015

Functional Genomics of the Aeromonas salmonicida Lipopolysaccharide O-Antigen and A-Layer from Typical and Atypical Strains.

Mar Drugs 2015 Jun 15;13(6):3791-808. Epub 2015 Jun 15.

Department of Microbiology, Faculty of Biology, University of Barcelona, Diagonal 643, Barcelona 08071, Spain.

The A. salmonicida A450 LPS O-antigen, encoded by the wbsalmo gene cluster, is exported through an ABC-2 transporter-dependent pathway. It represents the first example of an O-antigen LPS polysaccharide with three different monosaccharides in their repeating unit assembled by this pathway. Until now, only repeating units with one or two different monosaccharides have been described. Functional genomic analysis of this wbsalmo region is mostly in agreement with the LPS O-antigen structure of acetylated l-rhamnose (Rha), d-glucose (Glc), and 2-amino-2-deoxy-d-mannose (ManN). Between genes of the wbsalmo we found the genes responsible for the biosynthesis and assembly of the S-layer (named A-layer in these strains). Through comparative genomic analysis and in-frame deletions of some of the genes, we concluded that all the A. salmonicida typical and atypical strains, other than A. salmonicida subsp. pectinolytica strains, shared the same wbsalmo and presence of A-layer. A. salmonicida subsp. pectinolytica strains lack wbsalmo and A-layer, two major virulence factors, and this could be the reason they are the only ones not found as fish pathogens.
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http://dx.doi.org/10.3390/md13063791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4483657PMC
June 2015

Molecular and chemical analysis of the lipopolysaccharide from Aeromonas hydrophila strain AH-1 (Serotype O11).

Mar Drugs 2015 Apr 14;13(4):2233-49. Epub 2015 Apr 14.

Department of Microbiology, Faculty of Biology, University of Barcelona, Diagonal 643, 08071 Barcelona, Spain.

A group of virulent Aeromonas hydrophila, A. sobria, and A. veronii biovar sobria strains isolated from humans and fish have been described; these strains classified to serotype O11 are serologically related by their lipopolysaccharide (LPS) O-antigen (O-polysaccharide), and the presence of an S-layer consisting of multiple copies of a crystalline surface array protein with a molecular weight of 52 kDa in the form of a crystalline surface array which lies peripheral to the cell wall. A. hydrophila strain AH-1 is one of them. We isolated the LPS from this strain and determined the structure of the O-polysaccharide, which was similar to that previously described for another strain of serotype O11. The genetics of the O11-antigen showed the genes (wbO11 cluster) in two sections separated by genes involved in biosynthesis and assembly of the S-layer. The O11-antigen LPS is an example of an ABC-2-transporter-dependent pathway for O-antigen heteropolysaccharide (disaccharide) assembly. The genes involved in the biosynthesis of the LPS core (waaO11 cluster) were also identified in three different chromosome regions being nearly identical to the ones described for A. hydrophila AH-3 (serotype O34). The genetic data and preliminary chemical analysis indicated that the LPS core for strain AH-1 is identical to the one for strain AH-3.
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http://dx.doi.org/10.3390/md13042233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4413209PMC
April 2015

A BTP1 prophage gene present in invasive non-typhoidal Salmonella determines composition and length of the O-antigen of the lipopolysaccharide.

Mol Microbiol 2015 Apr 11;96(2):263-75. Epub 2015 Feb 11.

Centre for Immunology and Infection, Hull York Medical School and the Department of Biology, University of York, York, UK.

Salmonella Typhimurium isolate D23580 represents a recently identified ST313 lineage of invasive non-typhoidal Salmonellae (iNTS). One of the differences between this lineage and other non-iNTS S. Typhimurium isolates is the presence of prophage BTP1. This prophage encodes a gtrC gene, implicated in O-antigen modification. GtrC(BTP) (1) is essential for maintaining O-antigen length in isolate D23580, since a gtr(BTP) (1) mutant yields a short O-antigen. This phenotype can be complemented by gtrC(BTP) (1) or very closely related gtrC genes. The short O-antigen of the gtr(BTP) (1) mutant was also compensated by deletion of the BTP1 phage tailspike gene in the D23580 chromosome. This tailspike protein has a putative endorhamnosidase domain and thus may mediate O-antigen cleavage. Expression of the gtrC(BTP) (1) gene is, in contrast to expression of many other gtr operons, not subject to phase variation and transcriptional analysis suggests that gtrC is produced under a variety of conditions. Additionally, GtrC(BTP) (1) expression is necessary and sufficient to provide protection against BTP1 phage infection of an otherwise susceptible strain. These data are consistent with a model in which GtrC(BTP) (1) mediates modification of the BTP1 phage O-antigen receptor in lysogenic D23580, and thereby prevents superinfection by itself and other phage that uses the same O-antigen co-receptor.
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http://dx.doi.org/10.1111/mmi.12933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4413052PMC
April 2015

A Cronobacter turicensis O1 antigen-specific monoclonal antibody inhibits bacterial motility and entry into epithelial cells.

Infect Immun 2015 Mar 22;83(3):876-87. Epub 2014 Dec 22.

Department of Veterinary Science, Faculty of Veterinary Medicine, Ludwig-Maximilians-Universität München, Oberschleißheim, Germany.

Cronobacter turicensis is an opportunistic foodborne pathogen that can cause a rare but sometimes lethal infection in neonates. Little is known about the virulence mechanisms and intracellular lifestyle of this pathogen. In this study, we developed an IgG monoclonal antibody (MAb; MAb 2G4) that specifically recognizes the O1 antigen of C. turicensis cells. The antilipopolysaccharide antibody bound predominantly monovalently to the O antigen and reduced bacterial growth without causing cell agglutination. Furthermore, binding of the antibody to the O1 antigen of C. turicensis cells caused a significant reduction of the membrane potential which is required to energize flagellar rotation, accompanied by a decreased flagellum-based motility. These results indicate that binding of IgG to the O antigen of C. turicensis causes a direct antimicrobial effect. In addition, this feature of the antibody enabled new insight into the pathogenicity of C. turicensis. In a tissue culture infection model, pretreatment of C. turicensis with MAb 2G4 showed no difference in adhesion to human epithelial cells, whereas invasion of bacteria into Caco-2 cells was significantly inhibited.
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http://dx.doi.org/10.1128/IAI.02211-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333459PMC
March 2015

Defined single-gene and multi-gene deletion mutant collections in Salmonella enterica sv Typhimurium.

PLoS One 2014 9;9(7):e99820. Epub 2014 Jul 9.

Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, California, United States of America.

We constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (KanR) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (CamR) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a KanR cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a CamR cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099820PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4089911PMC
January 2016

An infection-relevant transcriptomic compendium for Salmonella enterica Serovar Typhimurium.

Cell Host Microbe 2013 Dec;14(6):683-95

Department of Microbiology, School of Genetics & Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin, Dublin 2, Ireland; Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK. Electronic address:

Bacterial transcriptional networks consist of hundreds of transcription factors and thousands of promoters. However, the true complexity of transcription in a bacterial pathogen and the effect of the environments encountered during infection remain to be established. We present a simplified approach for global promoter identification in bacteria using RNA-seq-based transcriptomic analyses of 22 distinct infection-relevant environmental conditions. Individual RNA samples were combined to identify most of the 3,838 Salmonella enterica serovar Typhimurium promoters in just two RNA-seq runs. Individual in vitro conditions stimulated characteristic transcriptional signatures, and the suite of 22 conditions induced transcription of 86% of all S. Typhimurium genes. We highlight the environmental conditions that induce the Salmonella pathogenicity islands and present a small RNA expression landscape of 280 sRNAs. This publicly available compendium of environmentally controlled expression of every transcriptional feature of S. Typhimurium constitutes a useful resource for the bacterial research community.
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http://dx.doi.org/10.1016/j.chom.2013.11.010DOI Listing
December 2013

The FUN of identifying gene function in bacterial pathogens; insights from Salmonella functional genomics.

Curr Opin Microbiol 2013 Oct 7;16(5):643-51. Epub 2013 Sep 7.

Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, United Kingdom.

The availability of thousands of genome sequences of bacterial pathogens poses a particular challenge because each genome contains hundreds of genes of unknown function (FUN). How can we easily discover which FUN genes encode important virulence factors? One solution is to combine two different functional genomic approaches. First, transcriptomics identifies bacterial FUN genes that show differential expression during the process of mammalian infection. Second, global mutagenesis identifies individual FUN genes that the pathogen requires to cause disease. The intersection of these datasets can reveal a small set of candidate genes most likely to encode novel virulence attributes. We demonstrate this approach with the Salmonella infection model, and propose that a similar strategy could be used for other bacterial pathogens.
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http://dx.doi.org/10.1016/j.mib.2013.07.009DOI Listing
October 2013

High-throughput comparison of gene fitness among related bacteria.

BMC Genomics 2012 May 30;13:212. Epub 2012 May 30.

University of California, Irvine, CA, USA.

Background: The contribution of a gene to the fitness of a bacterium can be assayed by whether and to what degree the bacterium tolerates transposon insertions in that gene. We use this fact to compare the fitness of syntenic homologous genes among related Salmonella strains and thereby reveal differences not apparent at the gene sequence level.

Results: A transposon Tn5 derivative was used to construct mutants in Salmonella Typhimurium ATCC14028 (STM1) and Salmonella Typhi Ty2 (STY1), which were then grown in rich media. The locations of 234,152 and 53,556 integration sites, respectively, were mapped by sequencing. These data were compared to similar data available for a different Ty2 isolate (STY2) and essential genes identified in E. coli K-12 (ECO). Of 277 genes considered essential in ECO, all had syntenic homologs in STM1, STY1, and STY2, and all but nine genes were either devoid of transposon insertions or had very few. For three of these nine genes, part of the annotated gene lacked transposon integrations (yejM, ftsN and murB). At least one of the other six genes, trpS, had a potentially functionally redundant gene encoded elsewhere in Salmonella but not in ECO. An additional 165 genes were almost entirely devoid of transposon integrations in all three Salmonella strains examined, including many genes associated with protein and DNA synthesis. Four of these genes (STM14_1498, STM14_2872, STM14_3360, and STM14_5442) are not found in E. coli. Notable differences in the extent of gene selection were also observed among the three different Salmonella isolates. Mutations in hns, for example, were selected against in STM1 but not in the two STY strains, which have a defect in rpoS rendering hns nonessential.

Conclusions: Comparisons among transposon integration profiles from different members of a species and among related species, all grown in similar conditions, identify differences in gene contributions to fitness among syntenic homologs. Further differences in fitness profiles among shared genes can be expected in other selective environments, with potential relevance for comparative systems biology.
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http://dx.doi.org/10.1186/1471-2164-13-212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3487940PMC
May 2012

Humanized nonobese diabetic-scid IL2rgammanull mice are susceptible to lethal Salmonella Typhi infection.

Proc Natl Acad Sci U S A 2010 Aug 16;107(35):15589-94. Epub 2010 Aug 16.

Departments of Laboratory Medicine and Microbiology, University of Washington School of Medicine, Seattle, WA 98195, USA.

Salmonella enterica serovar Typhi, the cause of typhoid fever, is host-adapted to humans and unable to cause disease in mice. Here, we show that S. Typhi can replicate in vivo in nonobese diabetic (NOD)-scid IL2rgamma(null) mice engrafted with human hematopoietic stem cells (hu-SRC-SCID mice) to cause a lethal infection with pathological and inflammatory cytokine responses resembling human typhoid. In contrast, S. Typhi does not exhibit net replication or cause illness in nonengrafted or immunocompetent control animals. Screening of transposon pools in hu-SRC-SCID mice revealed both known and previously unknown Salmonella virulence determinants, including Salmonella Pathogenicity Islands 1, 2, 3, 4, and 6. Our observations indicate that the presence of human immune cells allows the in vivo replication of S. Typhi in mice. The hu-SRC-SCID mouse provides an unprecedented opportunity to gain insights into S. Typhi pathogenesis and devise strategies for the prevention of typhoid fever.
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http://dx.doi.org/10.1073/pnas.1005566107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2932584PMC
August 2010

The Aeromonas hydrophila wb*O34 gene cluster: genetics and temperature regulation.

J Bacteriol 2008 Jun 11;190(12):4198-209. Epub 2008 Apr 11.

Departamento Microbiología, Facultad Biología, Universidad Barcelona, Diagonal 645, 08071 Barcelona. Spain.

The Aeromonas hydrophila wb*(O34) gene cluster of strain AH-3 (serotype O34) was cloned and sequenced. This cluster contains genes necessary for the production of O34-antigen lipopolysaccharide (LPS) in A. hydrophila. We determined, using either mutation or sequence homology, roles for the majority of genes in the cluster by using the chemical O34-antigen LPS structure obtained for strain AH-3. The O34-antigen LPS export system has been shown to be a Wzy-dependent pathway typical of heteropolysaccharide pathways. Furthermore, the production of A. hydrophila O34-antigen LPS in Escherichia coli K-12 strains is dependent on incorporation of the Gne enzyme (UDP-N-acetylgalactosamine 4-epimerase) necessary for the formation of UDP-galactosamine in these strains. By using rapid amplification of cDNA ends we were able to identify a transcription start site upstream of the terminal wzz gene, which showed differential transcription depending on the growth temperature of the strain. The Wzz protein is able to regulate the O34-antigen LPS chain length. The differential expression of this protein at different temperatures, which was substantially greater at 20 degrees C than at 37 degrees C, explains the previously observed differential production of O34-antigen LPS and its correlation with the virulence of A. hydrophila serotype O34 strains.
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http://dx.doi.org/10.1128/JB.00153-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2446748PMC
June 2008

Molecular analysis of three Aeromonas hydrophila AH-3 (serotype O34) lipopolysaccharide core biosynthesis gene clusters.

J Bacteriol 2008 May 29;190(9):3176-84. Epub 2008 Feb 29.

Departamento Microbiología, Facultad Biología, Universidad Barcelona, Diagonal 645, 08071 Barcelona, Spain.

By the isolation of three different Aeromonas hydrophila strain AH-3 (serotype O34) mutants with an altered lipopolysaccharide (LPS) migration in gels, three genomic regions encompassing LPS core biosynthesis genes were identified and characterized. When possible, mutants were constructed using each gene from the three regions, containing seven, four, and two genes (regions 1 to 3, respectively). The mutant LPS core structures were elucidated by using mass spectrometry, methylation analysis, and comparison with the full core structure of an O-antigen-lacking AH-3 mutant previously established by us. Combining the gene sequence and complementation test data with the structural data and phenotypic characterization of the mutant LPSs enabled a presumptive assignment of all LPS core biosynthesis gene functions in A. hydrophila AH-3. The three regions and the genes contained are in complete agreement with the recently sequenced genome of A. hydrophila ATCC 7966. The functions of the A. hydrophila genes waaC in region 3 and waaF in region 2 were completely established, allowing the genome annotations of the two heptosyl transferase products not previously assigned. Having the functions of all genes involved with the LPS core biosynthesis and most corresponding single-gene mutants now allows experimental work on the role of the LPS core in the virulence of A. hydrophila.
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http://dx.doi.org/10.1128/JB.01874-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2347379PMC
May 2008

Mesophilic Aeromonas UDP-glucose pyrophosphorylase (GalU) mutants show two types of lipopolysaccharide structures and reduced virulence.

Microbiology (Reading) 2007 Aug;153(Pt 8):2393-2404

Departamento de Microbiología, Facultad de Biología, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain.

A mutation in galU that causes the lack of O34-antigen lipopolysaccharide (LPS) in Aeromonas hydrophila strain AH-3 was identified. It was proved that A. hydrophila GalU is a UDP-glucose pyrophosphorylase responsible for synthesis of UDP-glucose from glucose 1-phosphate and UTP. The galU mutant from this strain showed two types of LPS structures, represented by two bands on LPS gels. The first one (slow-migrating band in gels) corresponds to a rough strain having the complete core, with two significant differences: it lacks the terminal galactose residue from the LPS-core and 4-amino-4-deoxyarabinose residues from phosphate groups in lipid A. The second one (fast-migrating band in gels) corresponds to a deeply truncated structure with the LPS-core restricted to one 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and three l-glycero-d-manno-heptose residues. galU mutants in several motile mesophilic Aeromonas strains from serotypes O1, O2, O11, O18, O21 and O44 were also devoid of the O-antigen LPS. The galU mutation reduced to less than 1 % the survival of these Aeromonas strains in serum, decreased the ability of these strains to adhere and reduced by 1.5 or 2 log units the virulence of Aeromonas serotype O34 strains in a septicaemia model in either fish or mice. All the changes observed in the galU mutants were rescued by the introduction of the corresponding single wild-type gene.
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http://dx.doi.org/10.1099/mic.0.2007/006437-0DOI Listing
August 2007

Non-structural flagella genes affecting both polar and lateral flagella-mediated motility in Aeromonas hydrophila.

Microbiology (Reading) 2007 Apr;153(Pt 4):1165-1175

Departamento de Microbiología, Facultad de Biología, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain.

An Aeromonas hydrophila AH-3 miniTn5 mutant unable to produce polar and lateral flagella was isolated, in which the transposon was inserted into a gene whose encoded protein was an orthologue of the Campylobacter jejuni motility accessory factor (Maf) protein. In addition to this gene, several other related genes were found in this cluster that was adjacent to the region 2 genes of the polar flagellum. Mutation of the A. hydrophila AH-3 maf-2, neuB-like, flmD or neuA-like genes resulted in non-motile cells that were unable to swim or swarm due to the absence of both polar and lateral flagella. However, both polar and lateral flagellins were present but were unglycosylated. Although the A. hydrophila AH-3 or Aeromonas caviae Sch3N genes did not hybridize with each other at the nucleotide level, the gene products were able to fully complement the mutations in either bacterium. Furthermore, well-characterized C. jejuni genes involved in flagella glycosylation (Cj1293, -1294 and -1317) were fully able to complement A. hydrophila mutants in the corresponding genes (flmA, flmB and neuB-like). It was concluded that the maf-2, neuB-like, flmD and neuA-like genes are involved in the glycosylation of both the polar and the lateral flagella in Aeromonas strains.
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http://dx.doi.org/10.1099/mic.0.2006/000687-0DOI Listing
April 2007

First description of nonmotile Vibrio vulnificus strains virulent for eels.

FEMS Microbiol Lett 2007 Jan;266(1):90-7

Departamento de Microbiología y Ecología, Universidad de Valencia, Burjassot, Valencia, Spain.

Nonmotile Vibrio vulnificus strains were isolated as pure cultures from body ulcers and internal organs of wild diseased European eels caught in a Mediterranean freshwater coastal lagoon. All 54 V. vulnificus isolates were nonmotile, indole-, ornithine decarboxilase-, mannitol- and cellobiose-positive, developed the opaque variant in culture, belonged to the O-antigenic serovar A and were highly virulent for eels by both intraperitoneal injection and immersion challenges. The nonmotile phenotype found in our V. vulnificus isolates was stable: nonmotile cells were always recovered from experimentally infected eels; no variation in the immobility of the V. vulnificus cells was observed for repeated subculture by daily passages on solid media, at different temperatures or incubation times and with or without magnesium sulfate. Many of the fla genes of Vibrio were present in the genome of the nonmotile strains (flaCDE and flaFBA for flagellins and flaH for the distal capping protein), although we observed by transmission electron microscopy that these V. vulnificus strains always lacked the polar flagellum. This is the first report on the existence of nonmotile wild-type V. vulnificus strains.
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http://dx.doi.org/10.1111/j.1574-6968.2006.00519.xDOI Listing
January 2007

A second galacturonic acid transferase is required for core lipopolysaccharide biosynthesis and complete capsule association with the cell surface in Klebsiella pneumoniae.

J Bacteriol 2007 Feb 1;189(3):1128-37. Epub 2006 Dec 1.

Departamento de Microbiología, Facultad de Biologia, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain.

The core lipopolysaccharide (LPS) of Klebsiella pneumoniae contains two galacturonic acid (GalA) residues, but only one GalA transferase (WabG) has been identified. Data from chemical and structural analysis of LPS isolated from a wabO mutant show the absence of the inner core beta-GalA residue linked to L-glycero-D-manno-heptose III (L,D-Hep III). An in vitro assay demonstrates that the purified WabO is able to catalyze the transfer of GalA from UDP-GalA to the acceptor LPS isolated from the wabO mutant, but not to LPS isolated from waaQ mutant (deficient in l,d-Hep III). The absence of this inner core beta-GalA residue results in a decrease in virulence in a capsule-dependent experimental mouse pneumonia model. In addition, this mutation leads to a strong reduction in cell-bound capsule. Interestingly, a K66 Klebsiella strain (natural isolate) without a functional wabO gene shows reduced levels of cell-bound capsule in comparison to those of other K66 strains. Thus, the WabO enzyme plays an important role in core LPS biosynthesis and determines the level of cell-bound capsule in Klebsiella pneumoniae.
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http://dx.doi.org/10.1128/JB.01489-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1797326PMC
February 2007

Role of Gne and GalE in the virulence of Aeromonas hydrophila serotype O34.

J Bacteriol 2007 Jan 10;189(2):540-50. Epub 2006 Nov 10.

Departamento Microbiología, Facultad Biología, Universidad Barcelona, Diagonal 645, 08071 Barcelona, Spain.

The mesophilic Aeromonas hydrophila AH-3 (serotype O34) strain shows two different UDP-hexose epimerases in its genome: GalE (EC 3.1.5.2) and Gne (EC 3.1.5.7). Similar homologues were detected in the different mesophilic Aeromonas strains tested. GalE shows only UDP-galactose 4-epimerase activity, while Gne is able to perform a dual activity (mainly UDP-N-acetyl galactosamine 4-epimerase and also UDP-galactose 4-epimerase). We studied the activities in vitro of both epimerases and also in vivo through the lipopolysaccharide (LPS) structure of A. hydrophila gne mutants, A. hydrophila galE mutants, A. hydrophila galE-gne double mutants, and independently complemented mutants with both genes. Furthermore, the enzymatic activity in vivo, which renders different LPS structures on the mentioned A. hydrophila mutant strains or the complemented mutants, allowed us to confirm a clear relationship between the virulence of these strains and the presence/absence of the O34 antigen LPS.
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http://dx.doi.org/10.1128/JB.01260-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1797372PMC
January 2007

Analysis of the lateral flagellar gene system of Aeromonas hydrophila AH-3.

J Bacteriol 2006 Feb;188(3):852-62

Departamento Microbiología, Facultad Biología, Universidad Barcelona, Diagonal 645, 08071 Barcelona, Spain.

Mesophilic Aeromonas strains express a polar flagellum in all culture conditions, and certain strains produce lateral flagella on semisolid media or on surfaces. Although Aeromonas lateral flagella have been described as a colonization factor, little is known about their organization and expression. Here we characterized the complete lateral flagellar gene cluster of Aeromonas hydrophila AH-3 containing 38 genes, 9 of which (lafA-U) have been reported previously. Among the flgLL and lafA structural genes we found a modification accessory factor gene (maf-5) that is involved in formation of lateral flagella; this is the first time that such a gene has been described for lateral flagellar gene systems. All Aeromonas lateral flagellar genes were located in a unique chromosomal region, in contrast to Vibrio parahaemolyticus, in which the analogous genes are distributed in two different chromosomal regions. In A. hydrophila mutations in flhAL, lafK, fliJL, flgNL, flgEL, and maf-5 resulted in a loss of lateral flagella and reductions in adherence and biofilm formation, but they did not affect polar flagellum synthesis. Furthermore, we also cloned and sequenced the A. hydrophila AH-3 alternative sigma factor sigma54 (rpoN); mutation of this factor suggested that it is involved in expression of both types of flagella.
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http://dx.doi.org/10.1128/JB.188.3.852-862.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1347325PMC
February 2006

Polar flagellum biogenesis in Aeromonas hydrophila.

J Bacteriol 2006 Jan;188(2):542-55

Departamento de Microbiología, Facultad de Biología, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain.

Mesophilic Aeromonas spp. constitutively express a single polar flagellum that helps the bacteria move to more favorable environments and is an important virulence and colonization factor. Certain strains can also produce multiple lateral flagella in semisolid media or over surfaces. We have previously reported 16 genes (flgN to flgL) that constitute region 1 of the Aeromonas hydrophila AH-3 polar flagellum biogenesis gene clusters. We identified 39 new polar flagellum genes distributed in four noncontiguous chromosome regions (regions 2 to 5). Region 2 contained six genes (flaA to maf-1), including a modification accessory factor gene (maf-1) that has not been previously reported and is thought to be involved in glycosylation of polar flagellum filament. Region 3 contained 29 genes (fliE to orf29), most of which are involved in flagellum basal body formation and chemotaxis. Region 4 contained a single gene involved in the motor stator formation (motX), and region 5 contained the three master regulatory genes for the A. hydrophila polar flagella (flrA to flrC). Mutations in the flaH, maf-1, fliM, flhA, fliA, and flrC genes, as well as the double mutant flaA flaB, all caused loss of polar flagella and reduction in adherence and biofilm formation. A defined mutation in the pomB stator gene did not affect polar flagellum motility, in contrast to the motX mutant, which was unable to swim even though it expressed a polar flagellum. Mutations in all of these genes did not affect lateral flagellum synthesis or swarming motility, showing that both A. hydrophila flagellum systems are entirely distinct.
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http://dx.doi.org/10.1128/JB.188.2.542-555.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1347287PMC
January 2006