Characterization and mapping of leaf rust resistance in four durum wheat cultivars.

Widening the genetic basis of leaf rust resistance is a primary objective of the global durum wheat breeding effort at the International Wheat and Maize Improvement Center (CIMMYT). Breeding programs in North America are following suit, especially after the emergence of new races of Puccinia triticina such as BBG/BP and BBBQD in Mexico and the United States, respectively. This study was conducted to characterize and map previously undescribed genes for leaf rust resistance in durum wheat and to develop reliable molecular markers for marker-assisted breeding. Four recombinant inbred line (RIL) mapping populations derived from the resistance sources Amria, Byblos, Geromtel_3 and Tunsyr_2, which were crossed to the susceptible line ATRED #2, were evaluated for their reaction to the Mexican race BBG/BP of P. triticina. Genetic analyses of host reactions indicated that leaf rust resistance in these genotypes was based on major seedling resistance genes. Allelism tests among resistant parents supported that Amria and Byblos carried allelic or closely linked genes. The resistance in Geromtel_3 and Tunsyr_2 also appeared to be allelic. Bulked segregant analysis using the Infinium iSelect 90K single nucleotide polymorphism (SNP) array identified two genomic regions for leaf rust resistance; one on chromosome 6BS for Geromtel_3 and Tunsyr_2 and the other on chromosome 7BL for Amria and Byblos. Polymorphic SNPs identified within these regions were converted to kompetitive allele-specific PCR (KASP) assays and used to genotype the RIL populations. KASP markers usw215 and usw218 were the closest to the resistance genes in Geromtel_3 and Tunsyr_2, while usw260 was closely linked to the resistance genes in Amria and Byblos. DNA sequences associated with these SNP markers were anchored to the wild emmer wheat (WEW) reference sequence, which identified several candidate resistance genes. The molecular markers reported herein will be useful to effectively pyramid these resistance genes with other previously marked genes into adapted, elite durum wheat genotypes.

High density mapping and haplotype analysis of the major stem-solidness locus SSt1 in durum and common wheat.

Breeding for solid-stemmed durum (Triticum turgidum L. var durum) and common wheat (Triticum aestivum L.) cultivars is one strategy to minimize yield losses caused by the wheat stem sawfly (Cephus cinctus Norton). Major stem-solidness QTL have been localized to the long arm of chromosome 3B in both wheat species, but it is unclear if these QTL span a common genetic interval. In this study, we have improved the resolution of the QTL on chromosome 3B in a durum (Kofa/W9262-260D3) and common wheat (Lillian/Vesper) mapping population. Coincident QTL (LOD = 94-127, R2 = 78-92%) were localized near the telomere of chromosome 3BL in both mapping populations, which we designate SSt1. We further examined the SSt1 interval by using available consensus maps for durum and common wheat and compared genetic to physical intervals by anchoring markers to the current version of the wild emmer wheat (WEW) reference sequence. These results suggest that the SSt1 interval spans a physical distance of 1.6 Mb in WEW (positions 833.4-835.0 Mb). In addition, minor QTL were identified on chromosomes 2A, 2D, 4A, and 5A that were found to synergistically enhance expression of SSt1 to increase stem-solidness. These results suggest that developing new wheat cultivars with improved stem-solidness is possible by combining SSt1 with favorable alleles at minor loci within both wheat species.

The abp locus of Streptococcus uberis encodes a protein homologous to polar amino acid and opine binding proteins of gram-negative bacteria.

A gene locus abp was identified immediately upstream of the CAMP factor gene cfu in Streptococcus uberis. An open reading frame capable of coding for a 277-residue protein was identified. On the basis of sequence characteristics, the abp gene product is potentially a polar amino acid and opine binding component of an ATP-binding cassette type (ABC-type) transport system similar to those of Gram-negative bacteria. This membrane protein is likely lipid modified at its amino terminus and was present in five S. uberis strains and one Streptococcus parauberis strain examined.

Cloning and analysis of duplicated rfbM and rfbK genes involved in the formation of GDP-mannose in Escherichia coli O9:K30 and participation of rfb genes in the synthesis of the group I K30 capsular polysaccharide.

The rfbO9 gene cluster, which is responsible for the synthesis of the lipopolysaccharide O9 antigen, was cloned from Escherichia coli O9:K30. The gnd gene, encoding 6-phosphogluconate dehydrogenase, was identified adjacent to the rfbO9 cluster, and by DNA sequence analysis the gene order gnd-rfbM-rfbK was established. This order differs from that described for other members of the family Enterobacteriaceae. Nucleotide sequence analysis was used to identify the rfbK and rfbM genes, encoding phosphomannomutase and GDP-mannose pyrophosphorylase, respectively. In members of the family Enterobacteriaceae, these enzymes act sequentially to form GDP-mannose, which serves as the activated sugar nucleotide precursor for mannose residues in cell surface polysaccharides. In the E. coli O9:K30 strain, a duplicated rfbM2-rfbK2 region was detected approximately 3 kbp downstream of rfbM1-rfbK1 and adjacent to the remaining genes of the rfbO9 cluster. The rfbM isogenes differed in upstream flanking DNA but were otherwise highly conserved. In contrast, the rfbK isogenes differed in downstream flanking DNA and in 3'-terminal regions, resulting in slight differences in the sizes of the predicted RfbK proteins. RfbMO9 and RfbKO9 are most closely related to CpsB and CpsG, respectively. These are isozymes of GDP-mannose pyrophosphorylase and phosphomannomutase, respectively, which are thought to be involved in the biosynthesis of the slime polysaccharide colanic acid in E. coli K-12 and Salmonella enterica serovar Typhimurium. An E. coli O-:K30 mutant, strain CWG44, lacks rfbM2-rfbK2 and has adjacent essential rfbO9 sequences deleted. The remaining chromosomal genes are therefore sufficient for GDP-mannose formation and K30 capsular polysaccharide synthesis. A mutant of E. coli CWG44, strain CWG152, was found to lack GDP-mannose pyrophosphorylase and lost the ability to synthesize K30 capsular polysaccharide. Wild-type capsular polysaccharide could be restored in CWG152, by transformation with plasmids containing either rfbM1 or rfbM2. Introduction of a complete rfbO9 gene cluster into CWG152 restored synthesis of both O9 and K30 polysaccharides. Consequently, rfbM is sufficient for the biosynthesis of GDP-mannose for both O antigen and capsular polysaccharide E. coli O9:K30. Analysis of a collection of serotype O8 and O9 isolates by Southern hybridization and PCR amplification experiments demonstrated extensive polymorphism in the rfbM-rfbK region.

Molecular analysis of the rfaD gene, for heptose synthesis, and the rfaF gene, for heptose transfer, in lipopolysaccharide synthesis in Salmonella typhimurium.

We report the analysis of three open reading frames of Salmonella typhimurium LT2 which we identified as rfaF, the structural gene for ADP-heptose:LPS heptosyltransferase II; rfaD, the structural gene for ADP-L-glycero-D-manno-heptose-6-epimerase; and part of kbl, the structural gene for 2-amino-3-ketobutyrate CoA ligase. A plasmid carrying rfaF complements an rfaF mutant of S. typhimurium; rfaD and kbl are homologous to and in the same location as the equivalent genes in Escherichia coli K-12. The RfaF (heptosyl transferase II) protein shares regions of amino acid homology with RfaC (heptosyltransferase I), RfaQ (postulated to be heptosyltransferase III), and KdtA (ketodeoxyoctonate transferase), suggesting that these regions function in heptose binding. E. coli contains a block of DNA of about 1,200 bp between kbl and rfaD which is missing from S. typhimurium. This DNA includes yibB, which is an open reading frame of unknown function, and two promoters upstream of rfaD (P3, a heat-shock promoter, and P2). Both S. typhimurium and E. coli rfaD genes share a normal consensus promoter (P1). We postulate that the yibB segment is an insertion into the line leading to E. coli from the common ancestor of the two genera, though it could be a deletion from the line leading to S. typhimurium. The G+C content of the rfaLKZYJI genes of both S. typhimurium LT2 and E. coli K-12 is about 35%, much lower than the average of enteric bacteria; if this low G+C content is due to lateral transfer from a source of low G+C content, it must have occurred prior to evolutionary divergence of the two genera.

Formation of the K30 (group I) capsule in Escherichia coli O9:K30 does not require attachment to lipopolysaccharide lipid A-core.

Escherichia coli K antigens (capsular polysaccharides) are divided into two broad classes, designated groups I and II, on the basis of a number of chemical, physical, and genetic criteria. Group I K antigens can be further subdivided on the basis of the absence (group IA) or presence (group IB) of amino sugars in the repeating unit of the K antigen. One criterion proposed for inclusion in group I is covalent linkage of the capsular polysaccharide to the lipid A-core of lipopolysaccharide (LPS). E. coli O9:K30 is a strain with a representative group IA K antigen. This organism synthesizes an LPS-associated low-molecular-weight form of K30 antigen which is called K(LPS). To determine the involvement of LPS lipid A-core in expression of the K30 capsular polysaccharide, E. coli K30/K-12 hybrid strains were constructed with mutations in the E. coli K-12 rfa locus, responsible for the biosynthesis of the LPS core oligosaccharide. These strains lack K(LPS), indicating that a full-length core is required for K(LPS) expression. However, formation of a K30 capsule was unaffected by rfa defects, indicating that attachment to lipid A-core is not an obligatory step for either export of high-molecular-weight capsular polysaccharide or maintenance of the capsular structure on the cell surface. Silver-stained tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of lipopolysaccharides from other E. coli K serotypes showed that all strains with group IB K antigens expressed some K(LPS). In contrast, some strains with group IA K antigens appear to lack K(LPS). Consequently, although association of group 1 K antigens with lipid A-core is common, it is not a universal marker for inclusion in group I.

Characterization of rcsB and rcsC from Escherichia coli O9:K30:H12 and examination of the role of the rcs regulatory system in expression of group I capsular polysaccharides.

In Escherichia coli K-12, RcsC and RcsB are thought to act as the sensor and effector components, respectively, of a two-component regulatory system which regulates expression of the slime polysaccharide colanic acid (V. Stout and S. Gottesman, J. Bacteriol. 172:659-669, 1990). Here, we report the cloning and DNA sequence of a 4.3-kb region containing rcsC and rcsB from E. coli O9:K30:H12. This strain does not produce colanic acid but does synthesize a K30 (group I) capsular polysaccharide. The rcsB gene from E. coli K30 (rcsBK30) is identical to the rcsB gene from E. coli K-12 (rcsBK-12). rcsCK30 has 16 nucleotide changes, resulting in six amino acid changes in the predicted protein. To examine the function of the rcs regulatory system in expression of the K30 capsular polysaccharide, chromosomal insertion mutations were constructed in E. coli O9:K30:H12 to independently inactivate rcsBK30 and the auxiliary positive regulator rcsAK30. Strains with these mutations maintained wild-type levels of K30 capsular polysaccharide expression and still produced a K30 capsule, indicating that the rcs system is not essential for expression of low levels of the group I capsular polysaccharide in lon+ E. coli K30. However, K30 synthesis is increased by introduction of a multicopy plasmid carrying rcsBK30. K30 polysaccharide expression is also markedly elevated in an rcsBK30-dependent fashion by a mutation in rcsCK30, suggesting that the rcs system is involved in high levels of synthesis. To determine whether the involvement of the rcs system in E. coli K30 expression is typical of group I (K antigen) capsules, multicopy rcsBK30 was introduced into 22 additional strains with structurally different group I capsules. All showed an increase in mucoid phenotype, and the polysaccharides produced in the presence and absence of multicopy rcsBK30 were examined. It is has been suggested that E. coli strains with group I capsules can be subdivided based on K antigen structure. For the first time, we show that strains with group I capsules can also be subdivided by the ability to produce colanic acid. Group IA contains capsular polysaccharides (including K30) with repeating-unit structures lacking amino sugars, and expression of group IA capsular polysaccharides is increased by multicopy rcsBK30. Group IB capsular polysaccharides all contain amino sugars. In group IB strains, multicopy rcsBK30 activates synthesis of colanic acid.

The rfaC gene of Salmonella typhimurium. Cloning, sequencing, and enzymatic function in heptose transfer to lipopolysaccharide.

We have cloned a gene from a Salmonella typhimurium with the ability to complement the rfaC mutation (heptose-deficient lipopolysaccharide, sensitivity to rough-specific bacteriophages, and susceptibility to hydrophobic antibiotics). A 1018-base pair EcoRV-Tth111I fragment, subcloned into the pBluescriptKS+ vector to yield pKZ103, retains complementing activity. Nucleotide sequencing revealed an open reading frame corresponding to a protein of 317 amino acids (M(r) approximately 35,100). The plasmid pKZ103, which has a properly aligned T7 promoter, can overexpress a protein of M(r) = 31,000 when T7 RNA polymerase is supplied. An in vitro system was established for analysis of heptose addition to the precursor [4'-32P](KDO)2-IVA (Brozek, K. A., Hosaka, K., Robertson, A. D., and Raetz, C. R. H. (1989) J. Biol. Chem, 264, 6956-6966). Soluble fractions from wild-type or heptose-deficient rfa mutants were tested for their ability to convert [4'-32P](KDO)2-IVA to more polar substances. In wild-type extracts, these conversions required addition of ATP or ADP-heptose. In extracts of rfaC-, rfaD-, or rfaE-deficient strains, no polar products were observed with ATP. ADP-heptose restored synthesis in rfaD and rfaE but not rfaC extracts, indicating that rfaD and rfaE are involved in ADP-heptose formation. When the cloned rfaC gene was introduced into an rfaC-deficient mutant, extracts from such cells regained the ability to metabolize [4'-32P](KDO)2-IVA, showing that rfaC encodes the enzyme that attaches the proximal heptose to lipopolysaccharide.

The rcsA gene of Escherichia coli O9:K30:H12 is involved in the expression of the serotype-specific group I K (capsular) antigen.

Escherichia coli produces two distinct types of capsular polysaccharide (designated groups I and II), which are distinguished by chemical, physical, and genetic characteristics. The K30 capsular antigen is a member of the group I, or heat-stable, capsules. We have cloned rcsA from E. coli O9:K30 and determined the nucleotide sequence. The rcsAK30 sequence is virtually identical to the rcsAK-12 sequence (V. Stout, A. Torres-Cabassa, M. R. Maurizi, D. Gutnick, and S. Gottesman, J. Bacteriol. 173:1738-1747, 1991). RcsAK-12 is a transcriptional activator involved in expression of the extracellular polysaccharide colanic acid in E. coli K-12. rcsAK30 complemented an rcsAK-12 mutation and activated colanic acid synthesis in E. coli K-12 strains. However, in E. coli K30, increasing the levels of RcsA by introducing multicopy rcsAK30 or a Lon mutation resulted in elevated synthesis of the K30 capsular polysaccharide; no colanic acid was detected. E. coli K-12 strains in which the chromosomal his region was replaced by that from E. coli K30 were able to synthesize K30 capsular polysaccharide. These K-12/K30 hybrid strains did not produce colanic acid, suggesting that the genes for synthesis of colanic acid and the K30 capsular polysaccharide may be allelic. rcsA sequences were also detected in the group II strains E. coli K1 and K5. Introduction of rcsAK30 into group II strains resulted in activation of colanic acid biosynthesis rather than the group II capsule. Given the role of RcsA in other members of the family Enterobacteriaceae, our results provide further evidence that this protein may be a relatively widespread regulatory component for the synthesis of enterobacterial extracellular polysaccharides.

Cloning, characterization, and DNA sequence of the rfaLK region for lipopolysaccharide synthesis in Salmonella typhimurium LT2.

We have cloned and sequenced the rfaL and rfaK genes for lipopolysaccharide synthesis in Salmonella typhimurium LT2 on a 4.28-kb HindIII fragment from the previously described R' factor pKZ3 (S. K. Kadam, A. Rehemtulla, and K. E. Sanderson, J. Bacteriol. 161:277-284, 1985). rfaL is thought to encode a component of the O-antigen ligase, and rfaK is believed to encode the N-acetylglucosamine transferase. The genes were identified by the loss of complementation of prototype rfaL and rfaK mutations after Tn1000 mutagenesis. Translation of the nucleotide sequence predicted sizes of 45.9 and 43.1 kDa for the rfaL and rfaK gene products, respectively. Hydropathy analysis of the rfaL product suggested that it was an integral membrane protein. A third gene, rfaZ, was found to be an 808-bp open reading frame on the pyrE side of rfaK. Insertions into rfaZ reduced rfaK complementation, suggesting cotranscription in the pyrE-cysE direction. The rfaL gene is transcribed in the opposite direction in a separate operon which may also include rfaC. An incomplete open reading frame with homology to an Escherichia coli gene in the same region, rfaY, was found on the pyrE side of rfaZ. Complementation studies with Tn1000 insertions in rfaL showed that rfaL446 and rfaL447 are allelic. With the cloning of the rfaL and -K genes, the order of genes within the rfa cluster at 79 units on the linkage map was found to be cysE-rfaDFCLKZYJIBG-pyrE.

The compositional analysis of bacterial extracellular polysaccharides by high-performance anion-exchange chromatography.

A high-performance liquid chromatography (HPLC) method with pulsed-amperometric detection (PAD) was developed for the compositional analysis of the acidic, neutral, and basic monosaccharides recovered from the acid hydrolysis of bacterial cell wall polysaccharides. This HPLC-PAD method involved the chromatography of the acid hydrolysis products on a CarboPac PA-1 anion-exchange column of pellicular resin, with PAD detection following postcolumn addition of alkali. Complete resolution of a mixture of 19 monosaccharides, comprising 9 neutral, 3 basic, and 7 acidic sugars, frequently found in bacterial polysaccharides was achieved within 60 min by the system. The presence of amino acids in the mixture was shown not to affect the analysis. This protocol was applied to the compositional analysis of 2 extracellular polysaccharides produced by Escherichia coli, colanic acid, and K30 antigen, which share constituent monosaccharides. The overproduction of extracellular polysaccharide in E. coli CWG56 was shown to be a consequence of deregulation of K30 biosynthesis and not of coexpression of an additional polymer.

Electrotransformation in Salmonella typhimurium LT2.

Electroporation gives high efficiency of transformation in Salmonella typhimurium LT2, yielding 10(8)-10(9) electrotransformants per microgram of pBR322 DNA. Lipopolysaccharide (LPS) composition has little influence on electrotransformation efficiency by electroporation, unlike Ca2+ shock methods, which give ca. 10(6) transformants/microgram DNA with strains with Rc or Rd2 LPS, 10(4) transformants with most smooth and rough strains, and 10(2) transformants with strains with Re LPS. Thus cell envelope properties are less crucial in electrotransformation than in Ca2+ shock methods. The reciprocal restriction barrier between Escherichia coli K-12 and S. typhimurium LT2 reduces electrotransformation by ca. 100-fold, but host-restriction mutants reduce or eliminate the barrier.

Transformation of Salmonella typhimurium with plasmid DNA: differences between rough and smooth strains.

Lipopolysaccharide-defective mutants of Salmonella typhimurium were transformed by plasmid DNA with a Ca2+ treatment method. Only those mutants with an Rc or Rd2 chemotype, due to galE or rfaF mutations, respectively, gave efficiencies greater than 10(5) transformants per microgram of DNA, frequencies 8- to 630-fold higher than with smooth strains or other rough mutants.

Derepression of F factor function in Salmonella typhimurium.

In Salmonella typhimurium LT2 the F factor of Escherichia coli K-12 replicates normally but is repressed; Flac+ cells give no visible lysis on solid media with male-specific phages, low frequency transfer of Flac+ (0.001-0.007 per donor cell), few f2 infective centers (0.002-0.006 per cell), and they propagate male-specific phages to low titers. Thus they display a Fin+ (fertility inhibition) phenotype. This repression, owing to pSLT, a 60 Mdal plasmid normally resident in S. typhimurium, was circumvented by the following materials: (i) Flac+ plasmids from E. coli with mutations in finP or traO; (ii) a S. typhimurium line which had been cured of pSLT; (iii) pKZl, a KmR plasmid in the same Inc group as pSLT, which caused expulsion of pSLT and made Fin- lines; (iv) F-Fin- mutants which originated spontaneously and which are present in most Hfr strains of S. typhimurium. Strains which are derepressed for F function by the above methods give visible lysis on solid media with male-specific phages, ca. 1.0 Lac+ recombinants per donor cell in conjugal transfer, ca. 0.82 f2 infective centers per cell, over 80% of cells with visible F pili, and propagation of male-specific phages to high titer. These data confirm earlier observations that pSLT represses F by the FinOP system. In addition, it shows that there is no other mechanism which represses F function in S. typhimurium. If donor function is derepressed by one of the above methods, and if rough recipient strains are used, F-mediated conjugation in S. typhimurium LT2 is as efficient as in E. coli K-12.