The innovation of the symbiosome has enhanced the evolutionary stability of nitrogen fixation in legumes

Nitrogen-fixing symbiosis is globally important in ecosystem functioning and agriculture, yet the evolutionary history of nodulation remains the focus of considerable debate. Recent evidence suggesting a single origin of nodulation followed by massive parallel evolutionary losses raises questions about why a few lineages in the N2 -fixing clade retained nodulation and diversified as stable nodulators, while most did not. Within legumes, nodulation is restricted to the two most diverse subfamilies, Papilionoideae and Caesalpinioideae, which show stable retention of nodulation across their core clades. We characterize two nodule anatomy types across 128 species in 56 of the 152 genera of the legume subfamily Caesalpinioideae: fixation thread nodules (FTs), where nitrogen-fixing bacteroids are retained within the apoplast in modified infection threads, and symbiosomes, where rhizobia are symplastically internalized in the host cell cytoplasm within membrane-bound symbiosomes (SYMs). Using a robust phylogenomic tree based on 997 genes from 147 Caesalpinioideae genera, we show that losses of nodulation are more prevalent in lineages with FTs than those with SYMs. We propose that evolution of the symbiosome allows for a more intimate and enduring symbiosis through tighter compartmentalization of their rhizobial microsymbionts, resulting in greater evolutionary stability of nodulation across this species-rich pantropical legume clade

High Density Microarray Analysis Reveals New Insights into Genetic Footprints of Listeria monocytogenes Strains Involved in Listeriosis Outbreaks

Listeria monocytogenes, a foodborne bacterial pathogen, causes invasive and febrile gastroenteritis forms of listeriosis in humans. Both invasive and febrile gastroenteritis listeriosis is caused mostly by serotypes 1/2a, 1/2b and 4b strains. The outbreak strains of serotype 1/2a and 4b could be further classified into several epidemic clones but the genetic bases for the diverse pathophysiology have been unsuccessful. DNA microarray provides an important tool to scan the entire genome for genetic signatures that may distinguish the L. monocytogenes strains belonging to different outbreaks. We have designed a pan-genomic microarray chip (Listeria GeneChip) containing sequences from 24 L. monocytogenes strains. The chip was designed to identify the presence/absence of genomic sequences, analyze transcription profiles and identify SNPs. Analysis of the genomic profiles of 38 outbreak strains representing 1/2a, 1/2b and 4b serotypes, revealed that the strains formed distinct genetic clusters adhering to their serotypes and epidemic clone types. Although serologically 1/2a and 1/b strains share common antigenic markers microarray analysis revealed that 1/2a strains are further apart from the closely related 1/2b and 4b strains. Within any given serotype and epidemic clone type the febrile gastroenteritis and invasive strains can be further distinguished based on several genetic markers including large numbers of phage genome, and intergenic sequences. Our results showed that the microarray-based data can be an important tool in characterization of L. monocytogenes strains involved in both invasive and gastroenteritis outbreaks. The results for the first time showed that the serotypes and epidemic clones are based on extensive pan-genomic variability and the 1/2b and 4bstrains are more closely related to each other than the 1/2a strains. The data also supported the hypothesis that the strains causing these two diverse outbreaks are genotypically different and this finding might be important in understanding the pathophysiology of this organism

Genomic characterization of Listeria monocytogenes strains involved in a multistate listeriosis outbreak associated with cantaloupe in US.

A multistate listeriosis outbreak associated with cantaloupe consumption was reported in the United States in September, 2011. The outbreak investigation recorded a total of 146 invasive illnesses, 30 deaths and one miscarriage. Subtyping of the outbreak associated clinical, food and environmental isolates revealed two serotypes (1/2a and 1/2b) and four pulsed-field gel electrophoresis two-enzyme pattern combinations I, II, III, and IV, including one rarely seen before this outbreak. A DNA-microarray, Listeria GeneChip®, developed by FDA from 24 Listeria monocytogenes genome sequences, was used to further characterize a representative sample of the outbreak isolates. The microarray data (in the form of present or absent calls of specific DNA sequences) separated the isolates into two distinct groups as per their serotypes. The gene content of the outbreak-associated isolates was distinct from that of the previously-reported outbreak strains belonging to the same serotypes. Although the 1/2b outbreak associated isolates are closely related to each other, the 1/2a isolates could be further divided into two distinct genomic groups, one represented by pattern combination I strains and the other represented by highly similar pattern combinations III and IV strains. Gene content analysis of these groups revealed unique genomic sequences associated with these two 1/2a genovars. This work underscores the utility of multiple approaches, such as serotyping, PFGE and DNA microarray analysis to characterize the composition of complex polyclonal listeriosis outbreaks

The organization of the genomic region containing the <i>lmo</i>0737 to <i>lmo0739</i> cassette that is present only in the <i>L. monocytogenes</i> serotypes IVb-v1 and 1/2a strains, confirmed by microarray analysis.

<p>The organization of the genomic region containing the <i>lmo</i>0737 to <i>lmo0739</i> cassette that is present only in the <i>L. monocytogenes</i> serotypes IVb-v1 and 1/2a strains, confirmed by microarray analysis.</p

Genomic Characterization of Novel <i>Listeria monocytogenes</i> Serotype 4b Variant Strains

<div><p>Over 90% of the human listeriosis cases are caused by <i>Listeria monocytogenes</i> serotypes 1/2a, 1/2b and 4b strains. As an alternative to antigen-antibody based serotyping, a PCR-based method for serogrouping has been developed and validated. In this communication, we report an in-depth analysis of five 4b variant strains, four clinical isolates from Australia and one environmental isolate from USA. Although these five strains were serotype 4b by classical serotyping method, the serogrouping PCR profiles of these strains show the presence of a 1/2a-3a specific amplicon in addition to the standard 4b-4d-4e specific amplicons. These strains were further analyzed by pulsed field gel electrophoresis, binary gene typing, multi-locus variable-number-tandem-repeat analysis and a high density pan-genomic <i>Listeria</i> microarray. Using these sub-typing results, the clinical isolates were grouped into two distinct genomic groups- one of which could be part of an unidentified outbreak. The microarray results when compared with our database of other 4b outbreak isolates indicated that the serotype 4b variant strains represent very different genotypic profiles than the known reported 4b outbreak strains representing major epidemic clones. The acquisition of serotype 1/2a gene clusters by the 4b variant strains appears to be independent in origin, spanning large areas of geographical and temporal space and may indicate predisposition of some 4b strains towards accepting DNA from related organisms.</p></div

Unique probe-sets in 1/2a and IVb-v1 <i>L. monocytogenes</i> strains.

<p>Unique probe-sets in 1/2a and IVb-v1 <i>L. monocytogenes</i> strains.</p

Multiplex PCR profiles of <i>L. monocytogenes</i> strains.

<p>Lanes: MW, Molecular weight markers. Lane 1: LS1 serotype 1/2a, Lane 2: LS402, serotype 4b; Lane3-7: serotype IVb-v1 strains LS542; LS642; LS643; LS644; LS645, respectively.</p

<i>L. monocytogenes</i> strains used in this study and their serotypic profiles.

<p>*Alternate designation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089024#pone.0089024-Huang1" target="_blank">[20]</a>.</p><p>**Antibody- based serotyping.</p><p>+/− indicates presence/absence of the band.</p

Scatter plots of the summarized Robust Multi-Array Averaging (RMA) intensities.

<p>A–C, between serotypes 4b (LS412) and IVb-v1 (LS642, A; LS644, B; LS645, C). D–E, between the same serotype, IVb-v1, isolated from Australia; from the different states (LS644 and LS645, D); from the same state (LS642 and LS644, E). F, between the same serotype, IVb-v1, isolated from the different countries (LS642; Australia and LS542; USA).</p

Hierarchical clustering based on the Robust Multi Array (RMA) analysis of the <i>L. monocytogenes</i> strains serotypes 4b (LS406, LS412) and IVb-v1 (LS542, LS642, LS643, LS644, LS645).

<p>Hierarchical clustering based on the Robust Multi Array (RMA) analysis of the <i>L. monocytogenes</i> strains serotypes 4b (LS406, LS412) and IVb-v1 (LS542, LS642, LS643, LS644, LS645).</p