6 Produce Degradation: Reaction Pathways and their Prevention
1.2.2 SAMPLE SEQUENCING
Sample sequencing is a low-cost but efficient approach to explore bacterial genomics.
Large-scale sample sequencing has been applied in the genome study of several
prokaryotic and eukaryotic microorganisms (Aguero et al., 2000; Sanchez et al.,
2001; Mittleider et al., 2002). Using E. carotovora subsp. atroseptica strain SCRI
1039, Bell et al. (2002) constructed a BAC library and identified two DNA fragments,
2B8 and 1C22, covering almost 200 kb of the bacterial genome. BLASTx/n searches
were conducted with onefold coverage sequences. Approximately 10% of the
E. carotovora subsp. atroseptica sequences showed the strongest similarity to those
of enterobacteria. The majority of these were found on 2B8. A number of sequences
similar to rhizobacterium genes were observed. Such genes were previously
unknown to Erwinia spp. or other enterobacteria. Conversely, strong matches in
1C22 were similar to adhesin, haemagglutanin, and haemolysin genes from mam-
malian pathogens such as Neisseria meningitidis and Pseudomonas aeruginosa and
the plant pathogen X. fastidiosa. Compared to other Erwinia species, genes other
than the targeted hrp and dsp genes, such as pel, peh, pec and hec genes, were
identified. Many of them were first identified in E. carotovora subsp. atroseptica.
1.2.3 SEQUENCES FROM rrn OPERONS
Sequences from the rrn locus, particularly the gene of small subunit ribosomal RNA
or 16S rDNA, is probably the most comprehensively collected DNA sequence among
all the bacterial genes from most bacterial species reported. Repeated targeting of
rDNA has yielded large 16S rDNA databases that provide the necessary reference
material for comparative sequence analysis and hence drawing reliable conclusions
on phylogeny. In fact, the reclassification and phylogenetics of soft rot erwinia
depend heavily on rRNA gene sequences (Kwon et al., 1997; Hauben et al., 1998;
Fessehaie et al., 2002).
Kwon et al. (1997) investigated the phylogenetic relationships of the type strains
of 16 Erwinia species by analyzing the 16S rDNA sequences using the neighbor-
joining method. Supported by moderate bootstrap values, the phylogenetic tree
shows that the genus Erwinia forms four phylogenetic clades or clusters. Among
them, cluster III consists of E. carotovora subspecies and E. chrysanthemi. The
Erwinia phylogeny clusters are, however, intermixed with members of other genera,
such as Escherichia coli, Klebsiella pneumoniae, and Serratia marcescens. Hauben
et al (1998) also analyzed the 16S rDNA sequences of 29 plant-associated strains
in Enterobacteriaceae, including Erwina. They proposed to include the soft rot
erwinia in the genus Pectobacterium, a genus name proposed over half of a century
ago (Waldee, 1945).
Although the validity of using a single gene for phylogenetic analysis has been
questioned, the universal presence of rRNA and its sequence conservation form the
basis to develop successful applications in bacterial identification (Jensen et al.,
1993). When the soft rot erwinia 16S rDNA sequences are aligned, differences at
the species and subspecies level are unambiguously displayed. As sequencing tech-
nology advances, sequence-base identification is becoming more practical and routine.