Rhizobium leguminosarum bv. trifolii rosR is required for interaction with clover, biofilm formation and adaptation to the environment

<p>Abstract</p> <p>Background</p> <p><it>Rhizobium leguminosarum </it>bv. <it>trifolii </it>is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants <it>Trifolium </it>spp. Bacterial surface polysaccharides are crucial for establishment of a successful symbiosis with legumes that form indeterminate-type nodules, such as <it>Trifolium</it>, <it>Pisum</it>, <it>Vicia</it>, and <it>Medicago </it>spp. and aid the bacterium in withstanding osmotic and other environmental stresses. Recently, the <it>R. leguminosarum </it>bv. <it>trifolii </it>RosR regulatory protein which controls exopolysaccharide production has been identified and characterized.</p> <p>Results</p> <p>In this work, we extend our earlier studies to the characterization of <it>rosR </it>mutants which exhibit pleiotropic phenotypes. The mutants produce three times less exopolysaccharide than the wild type, and the low-molecular-weight fraction in that polymer is greatly reduced. Mutation in <it>rosR </it>also results in quantitative alterations in the polysaccharide constituent of lipopolysaccharide. The <it>rosR </it>mutants are more sensitive to surface-active detergents, antibiotics of the beta-lactam group and some osmolytes, indicating changes in the bacterial membranes. In addition, the <it>rosR </it>mutants exhibit significant decrease in motility and form a biofilm on plastic surfaces, which differs significantly in depth, architecture, and bacterial viability from that of the wild type. The most striking effect of <it>rosR </it>mutation is the considerably decreased attachment and colonization of root hairs, indicating that the mutation affects the first stage of the invasion process. Infection threads initiate at a drastically reduced rate and frequently abort before they reach the base of root hairs. Although these mutants form nodules on clover, they are unable to fix nitrogen and are outcompeted by the wild type in mixed inoculations, demonstrating that functional <it>rosR </it>is important for competitive nodulation.</p> <p>Conclusions</p> <p>This report demonstrates the significant role RosR regulatory protein plays in bacterial stress adaptation and in the symbiotic relationship between clover and <it>R. leguminosarum </it>bv. <it>trifolii </it>24.2.</p

The chemical structure of the repeating unit of EPS produced by <i>R. leguminosarum</i> bv. <i>trifolii</i>[90].

<p>Abbreviations: Glc, glucose; GlcA, glucuronic acid; Gal, galactose; Ac, acetyl.</p

PssP2 Is a Polysaccharide Co-Polymerase Involved in Exopolysaccharide Chain-Length Determination in <i>Rhizobium leguminosarum</i>

<div><p>Production of extracellular polysaccharides is a complex process engaging proteins localized in different subcellular compartments, yet communicating with each other or even directly interacting in multicomponent complexes. Proteins involved in polymerization and transport of exopolysaccharide (EPS) in <i>Rhizobium leguminosarum</i> are encoded within the chromosomal Pss-I cluster. However, genes implicated in polysaccharide synthesis are common in rhizobia, with several homologues of <i>pss</i> genes identified in other regions of the <i>R. leguminosarum</i> genome. One such region is chromosomally located Pss-II encoding proteins homologous to known components of the Wzx/Wzy-dependent polysaccharide synthesis and transport systems. The <i>pssP2</i> gene encodes a protein similar to polysaccharide co-polymerases involved in determination of the length of polysaccharide chains in capsule and O-antigen biosynthesis. In this work, a mutant with a disrupted <i>pssP2</i> gene was constructed and its capabilities to produce EPS and enter into a symbiotic relationship with clover were studied. The <i>pssP2</i> mutant, while not altered in lipopolysaccharide (LPS), displayed changes in molecular mass distribution profile of EPS. Lack of the full-length PssP2 protein resulted in a reduction of high molecular weight EPS, yet polymerized to a longer length than in the RtTA1 wild type. The mutant strain was also more efficient in symbiotic performance. The functional interrelation between PssP2 and proteins encoded within the Pss-I region was further supported by data from bacterial two-hybrid assays providing evidence for PssP2 interactions with PssT polymerase, as well as glycosyltransferase PssC. A possible role for PssP2 in a complex involved in EPS chain-length determination is discussed.</p></div

Recommendations on the diagnosis of male infertility — genetic testing [Rekomendacje dotyczące diagnostyki genetycznej w niepłodności męskiej]

Male infertility is the cause of couples’ infertility in about 50% of cases. Current recommendations on the diagnosis and treatment of male infertility advance thorough medical history taking and physical examination, to provide the basis for further genetic evaluation. The extent of genetic testing itself depends on the semen analysis results, which allow the risk of inheritance of chromosomal aberrations to be determined and the root causes of habitual miscarriages to be explained.In azoospermia, once the type of microdeletion has been identified, a decision can be made as to whether a testicular biopsy is required to obtain sperm for the artificial reproductive technology (ART) procedure. The physical examination, genetic interview, and hormonal results are helpful in deciding which genetic tests to perform.Our research facilitates genetic testing in the diagnosis of male infertility

Analysis of the interaction between PssP2 and PssP in the <i>pssP2</i>::pKP2 mutant carrying the pQBP2his plasmid by a co-purification strategy.

<p>Samples of 40 µl of each fraction were separated by SDS-PAGE and visualized. (A) PssP is present in the fraction eluted from the resin, which equals to interaction between PssP and His₆-PssP2, that was bound to affinity resin via a His-tag. (B) Negative control verifying that co-purification of PssP is dependent on its interaction with the His-tagged PssP2. L, material loaded to the resin; W, last wash (10 resin volumes); E, elution (1 resin volume). Western blots for each protein are shown below the corresponding gels.</p

Strains, plasmids and oligonucleotide primers used in this work.

<p>Oligonucleotides were purchased from Genomed (Warsaw, Poland). Abbreviations: Str^r, streptomycin resistance; Rif^r, rifampin resistance; Km^r, kanamycin resistance; Tc^r, tetracycline resistance; Amp^r, ampicillin resistance; Gm^r, gentamicin resistance.</p><p>Strains, plasmids and oligonucleotide primers used in this work.</p

Putative homologues of PssP2 protein (586 aa) (ABD36550) identified through BLASTp searches.

<p>The database used above was the non-redundant UniProtKB/SwissProt. The multiple alignment of the above mentioned sequences is presented in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109106#pone.0109106.s001" target="_blank">Figure S1</a> (Supplementary data)</b>.</p><p>Putative homologues of PssP2 protein (586 aa) (ABD36550) identified through BLASTp searches.</p

Western immunoblot analysis with anti-His antibodies of subcellular localisation of His₆-PssP2 protein.

<p>Fractions analysed contained soluble proteins (SOL) and membrane proteins (TM) of the <i>pssP2</i>::pKP2 mutant and the complemented strain.</p

Summary of the highest scoring results from the HHpred search of the PssP2 protein against the PDB database.

<p>Summary of the highest scoring results from the HHpred search of the PssP2 protein against the PDB database.</p

Organization of genes in the Pss-II region, constructs used in the <i>pssP2</i> gene functional analyses and the results of PssP2 protein amino acid sequence analyses.

<p>A) Physical and genetic map of the <i>R. leguminosarum</i> bv. <i>trifolii</i> Pss-II region; genes encoding putative proteins similar to elements of the Wzx/Wzy-dependent polysaccharide polymerization pathway are indicated above the map; Rho-independent terminator predicted downstream <i>orf5</i> gene is marked with a <i>black rectangle</i>, promoters predicted between the <i>pssY</i> and <i>pssP2</i> genes are marked with <i>red rectangles</i>; B) Constructs used for integration mutagenesis of the <i>pssP2</i> gene (pKP2; <i>green bar</i>) and probing putative promoters identified upstream the <i>pssP2</i> (pMP-P2) and <i>pssY</i> (pMP-Y) genes (<i>blue bars</i>). Small <i>black rectangles</i> mark positions of primers used for amplification of promoter regions. <i>Red rectangles</i> below the pMP-P2 and pMP-Y constructs mark positions of identified promoters and the scores obtained for each predicted promoter. C) Genomic organization of the integration mutant <i>pssP2</i>::pKP2. Position of the p<i>lac</i> promoter (<i>red rectangle</i>) in the vector part and the direction of transcription from the promoter are shown. D) Sequence alignment of PssP2 and PssP proteins of <i>R. leguminosarum</i> bv. <i>trifoli</i> TA1. The alignment was produced in ClustalW and visualised by Alignment Viewer; amino acids were coloured according to their biochemical properties, thus the same colour means either identity or similarity, e.g. positively charged amino acids Arg and Lys are marked in red. E) Scheme of PssP2 topology and specific motifs found <i>in silico</i>. Blocks representing domains are aligned respective to the location in the polypeptide; TMS, transmembrane segment.</p