PMC:4236617 / 32928-36626 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4236617","sourcedb":"PMC","sourceid":"4236617","source_url":"http://www.ncbi.nlm.nih.gov/pmc/4236617","text":"Expression analysis of slp genes and comparison of surface-located Slp:s in silico\nIn an attempt to reveal which of the identified slp genes encoded the S-layer protein bands seen in the surface protein profiles of the strains (Figure 7), either an amino-terminal sequencing or a peptide mapping analysis was performed for the proteins, and the results were compared with the genomic sequence data. In this study, the major S-layer protein bands of the L. amylovorus isolates GRL 1112-GRL 1118 [28] were all shown to be encoded by slpA-like genes. The surface protein profiles of the strains DSM 16698 and DSM 20531T also revealed one major protein band, approximately 45 kDa in size (Figure 7), and, based on N-terminal sequencing, this represented the protein encoded by slpA. The presence of an S-layer on the surface of L. amylovorus DSM 16698 and DSM 20531T was thus confirmed in this study. Furthermore, the two additional surface protein bands of DSM 16698, approximately 50 kDa and 40 kDa in size, were found to represent the products of slpB- and slpC-like sequences, respectively. Of the three slpC-type genes present in the DSM 16698 genome, the plasmid-borne version, slpC3, was found to be expressed. Despite the presence of the SlpC-encoding gene on a plasmid, the SlpC band was invariably present in the SDS-PAGE profile of DSM 16698. In indirect immunofluorescence assays, SlpA and SlpB of DSM 16698 were identified on the bacterial surface as predicted. In contrast, SlpC remained undetectable, suggesting that the location of SlpC is not accessible to antibodies due to shielding by other cell envelope components (data not shown). The expressed slp genes of the L. amylovorus strains are highlighted in Figure 6.\nFigure 7 SDS-PAGE analysis of L. amylovorus strains. Intact cells of L. amylovorus DSM 16698 (lane 1), DSM 20531T (lane 2), GRL 1112 (lane 3), GRL 1114 (lane 4), GRL 1115 (lane 5), GRL 1116 (lane 6), GRL 1117 (lane 7) and GRL 1118 (lane 8) from 50 μl of overnight cultures (A600nm = 6.4) were boiled in standard Laemmli sample buffer (extracting surface proteins) and the supernatants were analyzed by standard SDS-PAGE in a 12% gel. Arrowheads indicate SlpA (44 kDa), SlpB (50 kDa) and SlpC (40 kDa) of L. amylovorus DSM 16698 (lane 1) and SlpA (61 kDa) and B (49 kDa) of GRL 1117 (lane 7). The designations and calculated molecular weights of the S-layer proteins present on the bacterial surface are summarized in Table 1, and the deduced amino acid sequences of these proteins are found in Additional file 1. The analysis of the Slp amino acid sequences revealed the typical features of Lactobacillus S-layer proteins, including a high predicted pI value (9.1-9.6) and a very low proportion of sulfur-containing amino acids [8]. A pairwise comparison of the amino acid sequence similarities of these proteins is shown in Table 2. An amino acid sequence alignment of these Slp:s and the major, surface-located S-layer proteins of L. acidophilus NCFM (SlpA, GenBank AAV42070) and L. crispatus JCM 5810 (CbsA, GenBank AF001313) is shown in Additional file 2. All the L. amylovorus S-layer proteins, with the exception of SlpC, display significant overall similarity to the L. acidophilus NCFM and L. crispatus Slp:s, with the signal peptides and the carboxy-terminal thirds of the sequences being particularly well conserved.\nTable 2 Amino acid sequence similarities between L. amylovorus S-layer proteins present on the bacterial surface 1Pairwise scores were calculated for each pair of sequences by calculating the number of identities in the best CLUSTALW alignment, and by dividing by the number of residues compared (gap positions were excluded).\n\n","divisions":[{"label":"Title","span":{"begin":0,"end":82}},{"label":"Figure caption","span":{"begin":1732,"end":2326}},{"label":"Table caption","span":{"begin":3362,"end":3697}}],"tracks":[{"project":"2_test","denotations":[{"id":"25070625-17544232-12189985","span":{"begin":495,"end":497},"obj":"17544232"},{"id":"25070625-23677442-12189986","span":{"begin":2764,"end":2765},"obj":"23677442"}],"attributes":[{"subj":"25070625-17544232-12189985","pred":"source","obj":"2_test"},{"subj":"25070625-23677442-12189986","pred":"source","obj":"2_test"}]},{"project":"MicrobeTaxon","denotations":[{"id":"T180","span":{"begin":453,"end":466},"obj":"1604"},{"id":"T181","span":{"begin":1135,"end":1144},"obj":"1604"},{"id":"T182","span":{"begin":1339,"end":1348},"obj":"1604"},{"id":"T183","span":{"begin":1406,"end":1415},"obj":"1604"},{"id":"T185","span":{"begin":2416,"end":2425},"obj":"2"},{"id":"T186","span":{"begin":2626,"end":2639},"obj":"1578"},{"id":"T187","span":{"begin":2968,"end":2987},"obj":"272621"},{"id":"T188","span":{"begin":3209,"end":3228},"obj":"272621"},{"id":"T189","span":{"begin":3017,"end":3029},"obj":"47770"},{"id":"T190","span":{"begin":3233,"end":3245},"obj":"47770"},{"id":"T191","span":{"begin":1681,"end":1694},"obj":"1604"},{"id":"T192","span":{"begin":3103,"end":3116},"obj":"1604"},{"id":"T193","span":{"begin":584,"end":601},"obj":"1604"},{"id":"T194","span":{"begin":606,"end":616},"obj":"1423723"},{"id":"T195","span":{"begin":852,"end":862},"obj":"1423723"},{"id":"T196","span":{"begin":824,"end":847},"obj":"1604"},{"id":"T197","span":{"begin":954,"end":963},"obj":"1604"},{"id":"T198","span":{"begin":584,"end":601},"obj":"695563"},{"id":"T199","span":{"begin":824,"end":847},"obj":"695563"},{"id":"T200","span":{"begin":954,"end":963},"obj":"695563"},{"id":"T201","span":{"begin":1135,"end":1144},"obj":"695563"},{"id":"T202","span":{"begin":1339,"end":1348},"obj":"695563"},{"id":"T203","span":{"begin":1406,"end":1415},"obj":"695563"},{"id":"T316","span":{"begin":1763,"end":1776},"obj":"1604"},{"id":"T317","span":{"begin":1802,"end":1825},"obj":"1604"},{"id":"T318","span":{"begin":2237,"end":2260},"obj":"1604"},{"id":"T319","span":{"begin":1836,"end":1846},"obj":"1423723"},{"id":"T320","span":{"begin":1802,"end":1825},"obj":"695563"},{"id":"T321","span":{"begin":2237,"end":2260},"obj":"695563"},{"id":"T184","span":{"begin":1439,"end":1448},"obj":"2"},{"id":"T331","span":{"begin":2416,"end":2425},"obj":"2"}],"namespaces":[{"prefix":"_base","uri":"http://purl.bioontology.org/ontology/NCBITAXON/"}],"attributes":[{"subj":"T180","pred":"source","obj":"MicrobeTaxon"},{"subj":"T181","pred":"source","obj":"MicrobeTaxon"},{"subj":"T182","pred":"source","obj":"MicrobeTaxon"},{"subj":"T183","pred":"source","obj":"MicrobeTaxon"},{"subj":"T185","pred":"source","obj":"MicrobeTaxon"},{"subj":"T186","pred":"source","obj":"MicrobeTaxon"},{"subj":"T187","pred":"source","obj":"MicrobeTaxon"},{"subj":"T188","pred":"source","obj":"MicrobeTaxon"},{"subj":"T189","pred":"source","obj":"MicrobeTaxon"},{"subj":"T190","pred":"source","obj":"MicrobeTaxon"},{"subj":"T191","pred":"source","obj":"MicrobeTaxon"},{"subj":"T192","pred":"source","obj":"MicrobeTaxon"},{"subj":"T193","pred":"source","obj":"MicrobeTaxon"},{"subj":"T194","pred":"source","obj":"MicrobeTaxon"},{"subj":"T195","pred":"source","obj":"MicrobeTaxon"},{"subj":"T196","pred":"source","obj":"MicrobeTaxon"},{"subj":"T197","pred":"source","obj":"MicrobeTaxon"},{"subj":"T198","pred":"source","obj":"MicrobeTaxon"},{"subj":"T199","pred":"source","obj":"MicrobeTaxon"},{"subj":"T200","pred":"source","obj":"MicrobeTaxon"},{"subj":"T201","pred":"source","obj":"MicrobeTaxon"},{"subj":"T202","pred":"source","obj":"MicrobeTaxon"},{"subj":"T203","pred":"source","obj":"MicrobeTaxon"},{"subj":"T316","pred":"source","obj":"MicrobeTaxon"},{"subj":"T317","pred":"source","obj":"MicrobeTaxon"},{"subj":"T318","pred":"source","obj":"MicrobeTaxon"},{"subj":"T319","pred":"source","obj":"MicrobeTaxon"},{"subj":"T320","pred":"source","obj":"MicrobeTaxon"},{"subj":"T321","pred":"source","obj":"MicrobeTaxon"},{"subj":"T184","pred":"source","obj":"MicrobeTaxon"},{"subj":"T331","pred":"source","obj":"MicrobeTaxon"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93ecad","default":true},{"id":"MicrobeTaxon","color":"#ec9393"}]}]}}