Essential Genes for
            <i>In Vitro</i>
            Growth of the Endophyte Herbaspirillum seropedicae SmR1 as Revealed by Transposon Insertion Site Sequencing

Rosconi, Federico; Vries, Stefan P. W. de; Baig, Abiyad; Fabiano, Elena; Grant, A.

doi:10.1128/aem.02281-16

Cited by 18 publications

(16 citation statements)

References 45 publications

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“…Another essential ncRNA was rnpB which is a component RNA of the ribonuclease P enzyme (RNase P) together with the RnpA protein (also identified as an essential gene in our study) functions in the processing of 4.5S RNA and tRNA precursor molecules in E. coli ( https://ecocyc.org/gene?orgid=ECOLI&id=EG30069 ). Similar to our findings, in a recent study, Rosconi et al (2016) found 22 tRNA genes and one 23S ribosomal RNA gene to be essential in Herbaspirillum seropedicae , an endophyte that colonizes crops like rice and maize, during in vitro growth in TY medium [ 38 ].…”

Section: Resultssupporting

confidence: 92%

Essential genome of Campylobacter jejuni

2017

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Background Campylobacter species are a leading cause of bacterial foodborne illness worldwide. Despite the global efforts to curb them, Campylobacter infections have increased continuously in both developed and developing countries. The development of effective strategies to control the infection by this pathogen is warranted. The essential genes of bacteria are the most prominent targets for this purpose. In this study, we used transposon sequencing (Tn-seq) of a genome-saturating library of Tn5 insertion mutants to define the essential genome of C. jejuni at a high resolution. ResultWe constructed a Tn5 mutant library of unprecedented complexity in C. jejuni NCTC 11168 with 95,929 unique insertions throughout the genome and used the genomic DNA of the library for the reconstruction of Tn5 libraries in the same (C. jejuni NCTC 11168) and different strain background (C. jejuni 81–176) through natural transformation. We identified 166 essential protein-coding genes and 20 essential transfer RNAs (tRNA) in C. jejuni NCTC 11168 which were intolerant to Tn5 insertions during in vitro growth. The reconstructed C. jejuni 81–176 library had 384 protein coding genes with no Tn5 insertions. Essential genes in both strain backgrounds were highly enriched in the cluster of orthologous group (COG) categories of ‘Translation, ribosomal structure and biogenesis (J)’, ‘Energy production and conversion (C)’, and ‘Coenzyme transport and metabolism (H)’.ConclusionComparative analysis among this and previous studies identified 50 core essential genes of C. jejuni, which can be further investigated for the development of novel strategies to control the spread of this notorious foodborne bacterial pathogen.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-4032-8) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 92%

Essential genome of Campylobacter jejuni

2017

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“…Seven bands with differential expression intensities were selected for identification by peptide mapping in a MALDI-TOF mass spectrometer ( Figure 3 ). Three bands that migrate very close in the low iron availability environment were analyzed as a single sample in the MALDI-TOF, and are collectively referred to as band E. As a control we included one band with similar intensity in both conditions, which was named band A and was shown to be the product of the gene Hsero_4295, an outer membrane porin identified recently as a candidate essential gene of H. seropedicae ( Rosconi et al, 2016 ). Band B, highly expressed in iron sufficient condition, was identified as the product of Hsero_2973, a homolog of sdhA , encoding the succinate dehydrogenase flavoprotein subunit that contains a Fe-S cluster ( McNeil et al, 2014 ).…”

Section: Resultsmentioning

confidence: 99%

Herbaspirillum seropedicae Differentially Expressed Genes in Response to Iron Availability

et al. 2018

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Herbaspirillum seropedicae Z67 is a nitrogen-fixing endophyte that colonizes many important crops. Like in almost all organisms, vital cellular processes of this endophyte are iron dependent. In order to efficiently acquire iron to fulfill its requirements, this bacterium produces the siderophores serobactins. However, the presence in its genome of many others iron acquisition genes suggests that serobactins are not the only strategy used by H. seropedicae to overcome metal deficiency. The aim of this work was to identify genes and proteins differentially expressed by cells growing in low iron conditions in order to describe H. seropedicae response to iron limitation stress. For this purpose, and by using a transcriptomic approach, we searched and identified a set of genes up-regulated when iron was scarce. One of them, Hsero_2337, codes for a TonB-dependent transporter/transducer present in the serobactins biosynthesis genomic locus, with an unknown function. Another TonB-dependent receptor, the one encoded by Hsero_1277, and an inner membrane ferrous iron permease, coded by Hsero_2720, were also detected. By using a proteomic approach focused in membrane proteins, we identified the specific receptor for iron-serobactin internalization SbtR and two non-characterized TonB-dependent receptors (coded by genes Hsero_1277 and Hsero_3255). We constructed mutants on some of the identified genes and characterized them by in vitro growth, biofilm formation, and interaction with rice plants. Characterization of mutants in gene Hsero_2337 showed that the TonB-dependent receptor coded by this gene has a regulatory role in the biosynthesis of serobactins, probably by interacting with the alternative sigma factor PfrI, coded by gene Hsero_2338. Plant colonization of the mutant strains was not affected, since the mutant strain normally colonize the root and aerial part of rice plants. These results suggest that the strategies used by H. seropedicae to acquire iron inside plants are far more diverse than the ones characterized in this work. In vivo expression studies or colonization competition experiments between the different mutant strains could help us in future works to determine the relative importance of the different iron acquisition systems in the interaction of H. seropedicae with rice plants.

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“…Beneficial bacteria need to not only cope with a range of environments and climatic conditions, but actively enhance plant growth under such conditions. In addition to colonizing the crop host, the bacteria need to compete with existing soil microbiota for resources and be safe for the environment (Rosconi et al, 2016). The bacterial inoculum may be applied in conjunction with chemical pesticides and fertilizers, so it is advantageous if it is able to survive and maintain plant growth promoting activity in the presence of these additions and must also be able to survive packaging, transport and storage before it is applied to a crop (Glick, 2015;Backer et al, 2018).…”

Section: Boosting Crop Yields Via Application Of Beneficial Bacteriamentioning

confidence: 99%

Application of Transposon Insertion Sequencing to Agricultural Science

2020

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Many plant-associated bacteria have the ability to positively affect plant growth and there is growing interest in utilizing such bacteria in agricultural settings to reduce reliance on pesticides and fertilizers. However, our capacity to utilize microbes in this way is currently limited due to patchy understanding of bacterial-plant interactions at a molecular level. Traditional methods of studying molecular interactions have sought to characterize the function of one gene at a time, but the slow pace of this work means the functions of the vast majority of bacterial genes remain unknown or poorly understood. New approaches to improve and speed up investigations into the functions of bacterial genes in agricultural systems will facilitate efforts to optimize microbial communities and develop microbe-based products. Techniques enabling high-throughput gene functional analysis, such as transposon insertion sequencing analyses, have great potential to be widely applied to determine key aspects of plant-bacterial interactions. Transposon insertion sequencing combines saturation transposon mutagenesis and high-throughput sequencing to simultaneously investigate the function of all the nonessential genes in a bacterial genome. This technique can be used for both in vitro and in vivo studies to identify genes involved in microbe-plant interactions, stress tolerance and pathogen virulence. The information provided by such investigations will rapidly accelerate the rate of bacterial gene functional determination and provide insights into the genes and pathways that underlie biotic interactions, metabolism, and survival of agriculturally relevant bacteria. This knowledge could be used to select the most appropriate plant growth promoting bacteria for a specific set of conditions, formulating crop inoculants, or developing crop protection products. This review provides an overview of transposon insertion sequencing, outlines how this approach has been applied to study plant-associated bacteria, and proposes new applications of these techniques for the benefit of agriculture.

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Essential Genes for In Vitro Growth of the Endophyte Herbaspirillum seropedicae SmR1 as Revealed by Transposon Insertion Site Sequencing

Cited by 18 publications

References 45 publications

Essential genome of Campylobacter jejuni

Essential genome of Campylobacter jejuni

Herbaspirillum seropedicae Differentially Expressed Genes in Response to Iron Availability

Application of Transposon Insertion Sequencing to Agricultural Science

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