20C2

Site of transposon insertion cloned as PstI fragment into pSP72. 
Sequenced in opposite orientations with JEP131 and JEP132,
from sites at opposing ends of the transposon, to give:

JEP131 782 bp

JEP132 795 bp
 
Composite sequence:

ATGTCGGGAT GACACAGCAA AAGCCGTGCT TGAGCATCAG GCGGATATGG
GAATTGCTTT TGACGGTGAT TTCGATCGTT GTTTCCTGTT TGATGAGAAG
GGCGCTTTCA TCGAGGGCTA CTATATCGTT GGCCTATTGG CAGAAGCTTT
CCTGCAAGCC AACCCTGGCT CCAAGATTAT TCATGATCCT CGTCTGTCAT
GGAATACCAT CGATATCGTT ACCCAAGCTG GTGGCATCCC TGTTATGTCC
AAAACGGGGC ATGCCTTTAT CAAGGAGCGC ATGCGCAAAG AAGATGCTAT
CTATGGTGGC GAGATGAGTG CGCACCATTA CTTCCGTGAT TTCGCTTATT
GTGACAGCGG TATGATTCCA TGGCTATTGG TAGCGGGTTT ACTTTCAGTT
AAAGGCAGTG TGTTGAGTCA GCTAGTGAAG GATCGTATGG AAGCGTATCC
AGCATCTGGT GAGATTAATC GCAAATTGCC TGATGCTAAA GCTGCCATTG
AGCGTGTTTT AGGCGTTTAC AAAGATGATG CCGTGGCTTT TGACATGACA
GATGGTATCA GCCTTGAATT CAATGAATGG CGTTTTAATC TGCGGACATC
GAATACTGAA CCTGTTGTAC GTTTGAATGT TGAGTCAAAA GGGGATGTTG
AATTGATGCA GAAAAAAACT AAAGAGATTC TGGCGCTCTT AGGCGGAGAA
TAACGATGAA AGAAATGTTA TTAGCCATCT ATGGTTACCG GGGTTTTATT
TGGAGCAGTA TTAAGCGGGA CTTTCAGTCC CG

TTACCAAACC AGCATGTTGG GAGCTTTATG GCTGGTATTG CAGCCGTTAG
CGATGATTTT GGTATATACA CTGGTATTCT CAGAAGTGAT GAAGGCACGG
ATGCCTGATA ATTCAGGTCC TTTTGCCTAT AGTATTTATC TTTGCTCTGG
GGTGCTGACG TGGGGATTAT TTACTGAAAT TCTTGATAAA AGCCAAAGTG
TCTTCATTAC TAATGCTAAC TTAATAAAAA AGCTCAGTTT CCCAAAAATT
TGCTTGCCGG TCATTGTGAC CGCGTCTGCG ATTTTAAACT TCGCCATTAT
ATTTAGCCTG TTTTTAATTT TTCTTGTTTT TACCGGAAAC TTTCCCGGGG
TTTTATTCTT CTCCGTCTTA CCCGTTTTGG CTCTGCAAGT TCTGTTCACC
GTCGGTCTGG GTATGACATT GGGCGTGATG AACGTTTTCT TCCGGGACGT
TGGGCAGTTT GTTGGCGTAT TATTGCAATT CTGGTTCTGG TTCACACCCA
TCGTTTATGT ATTAAATTCG CTACCTGAAT GGGCCAGAAC ATCGCTGATG
TACAATCCAA TGGCCCGAGT TATGCAAGCA TACCAGTCAA TATTTGCATA
CCAACAGCAA CCTAACTGGT ACTCATTATG GCCAGTATTG GTTCTGGCCA
TAATTTTCTG TGCGTTTGGT ATGAGATTAT TCAGGAAACA CGCTGCAGGC
ATGCAAGCTT CAGCTGCTCG AGTTCTATAG TGTCACCTAA ATCGTATGTG
TATGATACAT AAGGTTATGT ATTAATTGTA GCCGCGTTCT ACGAC


BLASTX with this sequence (full results here) gives as most significant hit :


>gi|3142211|dbj|BAA28324.1| (AB010150) integral membrane component of ABC-transporter
            [Escherichia coli]
          Length = 264

 Score =  416 bits (1070), Expect = e-116
 Identities = 213/260 (81%), Positives = 232/260 (88%)
 Frame = +1

Query: 706  MKEMLLAIYGYRGFIWSSIKRDFQSRYQTSMLGALWLVLQPLAMILVYTLVFSEVMKARM 885
            M+++L  IY YRGFIWSS+KRDFQ+RYQTSMLGALWLVLQPL+MILVYTLVFSEVMKARM
Sbjct: 1    MRDLLTTIYRYRGFIWSSVKRDFQARYQTSMLGALWLVLQPLSMILVYTLVFSEVMKARM 60

Query: 886  PDNSGPFAYSIYLCSGVLTWGLFTEILDKSQSVFITNANLIKKLSFPKICLPVIVTASAI 1065
            PDN+G FAYSIYLCSGVLTWGLFTE+LDK QSVFI NANLIKKLSFPKICLP+IVT SA+
Sbjct: 61   PDNTGSFAYSIYLCSGVLTWGLFTEMLDKGQSVFINNANLIKKLSFPKICLPIIVTLSAV 120

Query: 1066 LNFAIIFSLFLIFLVFTGNFPGVLFFSVLPVLALQVLFTVGLGMTLGVMNVFFRDVGQFV 1245
            LNFAIIFSLFLIF++ TGNFPG LF SV+PVL LQ+LF  GLGM LGVMNVFFRDVGQ V
Sbjct: 121  LNFAIIFSLFLIFIIVTGNFPGWLFLSVIPVLLLQILFAGGLGMILGVMNVFFRDVGQLV 180

Query: 1246 GVLLQFWFWFTPIVYVLNSLPEWARTSLMYNPMARVMQAYQSIFAYQQQPNWYSLWPVLV 1425
            GV LQFWFWFTPIVYVLNSLP WA+  +MYNPM R+MQ+YQSIFAY   PNWYSLWPVL 
Sbjct: 181  GVALQFWFWFTPIVYVLNSLPAWAKNLMMYNPMTRIMQSYQSIFAYHLAPNWYSLWPVLA 240

Query: 1426 LAIIFCAFGMRLFRKHAAGM 1485
            LAIIFC  G R+FRKHAA M
Sbjct: 241  LAIIFCVIGFRMFRKHAADM 260


and also


>gi|3930206|gb|AAC80270.1| (AF031957) phosphomannomutase [Shigella sonnei]
          Length = 456

 Score =  369 bits (946), Expect = e-101
 Identities = 179/230 (77%), Positives = 198/230 (85%)
 Frame = +2

Query: 2   CRDDTAKAVLEHQADMGIAFDGDFDRCFLFDEKGAFIEGYYIVGLLAEAFLQANPGSKII 181
           CRDDT  AV++H ADMGIAFDGDFDRCFLFDEKG FIEGYYIVGLLAEAFL+ NPG+KII
Sbjct: 225 CRDDTRNAVIKHGADMGIAFDGDFDRCFLFDEKGQFIEGYYIVGLLAEAFLEKNPGAKII 284

Query: 182 HDPRLSWNTIDIVTQAGGIPVMSKTGHAFIKERMRKEDAIYGGEMSAHHYFRDFAYCDSG 361
           HDPRLSWNT+D+VT AGG PVMSKTGHAFIKERMRKEDAIYGGEMSAHHYFRDFAYCDSG
Sbjct: 285 HDPRLSWNTVDVVTAAGGTPVMSKTGHAFIKERMRKEDAIYGGEMSAHHYFRDFAYCDSG 344

Query: 362 MIPWLLVAGLLSVKGSVLSQLVKDRMEAYPASGEINRKLPDAKAAIERVLGVYKDDAVAF 541
           MIPWLLVA L+ +KG  L +LV+DRM A+PASGEIN KL     AI RV   +  +A+A 
Sbjct: 345 MIPWLLVAELVCLKGKTLGELVRDRMAAFPASGEINSKLAQPVEAINRVEQHFSREALAV 404

Query: 542 DMTDGISLEFNEWRFNLRTSNTEPVVRLNVESKGDVELMQKKTKEILALL 691
           D TDGIS+ F +WRFNLRTSNTEPVVRLNVES+GDV LM++KTK IL LL
Sbjct: 405 DRTDGISMTFADWRFNLRTSNTEPVVRLNVESRGDVPLMEEKTKLILELL 454

The transposon is thus inserted within the coding sequence of a gene that encodes a homologue of an ABC transporter (wzm), with the adjacent gene being a homologue of a phosphomannomutase.

STRING analysis indicates that these two genes are often found together (see colour code) and are involved in LPS biosynthesis.

The most heterogeneous portion of LPS, O-antigens, have long been recognised as an important determinant of bacterial virulence in many bacteria (see for example this review by Rocchetta et al. on O-antigen biosynthesis in Pseudomonas aeruginosa).

The Serratia marcescens N28b O4 antigen gene cluster, that has been characterised by Saigi et al., contains a wzm homologue, similar, but not identical to the predicted protein product of the gene disrupted in 20C2 (see BLAST2 results). The O4 antigen gene cluster was not reported to contain a phosphomannomutase.

In the Pseudomonas aeruginosa Community Annotation Project, one can see that the closest relative of the 20C2 protein in P. aeruginosa (PA5451) (see BLASTP search results) has been characterised as a membrane subunit of an A-band LPS efflux transporter, with the upstream gene being a phosphomannose isomerase.

P. aeruginosa coexpresses two distinct lipopolysaccharide (LPS) molecules: B band a serospecific LPS, and A band the common LPS antigen (see Rocchetta & Lam 1997).