Comparative Analysis of L monocytogenes with Available Genome Sequences

Analysis of the available sequences revealed that the Listeria genomes are very homogeneous in size ranging from 2893 to 3,011 kb with conserved general features summarized in Table 3.1. If integrated prophages are not considered, the L. innocua genome, which contains five integrated prophages accounting for 216.8 kb, has the smallest size of 2,794 kb containing 2,679 protein-encoding genes (Table 3.1.). Based on the 16S rRNA sequence, the genus Listeria is classified into the group of low GC Gram positive bacteria closely related to Bacillaceae. Another means to compare organisms is based on gene content of their genomes and on the distribution of the highest similarity score of each protein or on the number of bidirectional best hits (BDBH). In 2001, when the genes predicted in the L. monocytogenes strain EGDe were first annotated, 318 genes were scored as coding proteins without similarity to any other protein in the public sequence libraries or as showing similarity only to other L. monocytogenes proteins. Since then, an enormous amount of new sequences has been added to these public databases. Thus, it was expected that these scores might have changed. In order to be able to compare the results we had gotten in 2001 with the situation 5 years later, we re-analyzed the similarity scores of all predicted protein sequences of L. monocytogenes EGDe to proteins present in the public databases. We recorded the first six non-Listeria BLASTp hits in the NCBI-NR protein sequence database with each of the EGDe proteins. This database contains protein sequences from over 500 complete or unfinished bacterial genome sequences. Surprisingly, out of the originally 318 orphan genes, only 60 protein sequences had now a similar sequence among this tremendous amount of new protein

Table 3.1. General features of published Listeria genome sequences.

L. monocyto

L. monocyto-

L .monocyto-

L.monocytogenes

L.innocua

genes EGDe

genes F6854

genes F2365

H7858(4b)c

CLIP11262

(1/2a)

(1/2a)c

(4b)

(6a)

Size of the

2 944 528

~ 2953 211

2 905 310

~ 2 893 921

3 011209

chromosome

(bp)

G+C content

38

37.8

38

38

37.4

(%)

G+C content

38.4

38.5

38.5

38.4

38.0

protein-coding

genes (%)

Total number

2853

2973

2847

3024

2973

of CDSa

Percentage

89.2%

90.3%

88.4%

89.5%

89.1%

coding

Number of

1

3

2

2

5

prophage

regions

Monocins

1

1

1

1

1

Plasmid

-

-

-

1 (94 CDS)

1 (79 CDS)

Number of

61

97

51

69

78

strain-specific

genesb

Number of

1 (Tn916 like)

-

-

-

-

transposons

Number of

6

6

6

6

6

rRNA operons

Number of

67

67c

67

65c

66

tRNA genes

aCDS = coding sequence bexcept prophage genes c Draft genome sequence (eightfold coverage without gap closure)

aCDS = coding sequence bexcept prophage genes c Draft genome sequence (eightfold coverage without gap closure)

sequences. Thus 258 genes still encode proteins that are orphans. This result strongly supports the view that these genes are specific of the genus Listeria.

As horizontal gene transfer may play a considerable role in the adaptation of a bacterium to its specific niche(s), we have compared the occurrence of best BLASTp and phylogeny. In Figure 3.1., the distribution of best BLASTp hits among bacterial genera or higher order taxonomic groups is presented. In agreement with the phylogeny, the largest number of best hits was detected among Bacillaceae, with 1,523 genes, which represent 58% of those that encode a protein that has a similarity hit with a protein in the public database. These proteins constitute the core genome of this group of bacteria, encoding mainly functions like the transcription and translation machinery and metabolic pathways. The remaining genes, the flexible gene pool, are probably responsible for the adaptation of these bacteria to their different environments and niches where they are found. When analyzing the distribution of the best hits of these

OBacillus - 1523

□ Clostridium- 235

□ Lactobacillus -169

■ Streptococcus lactococcus - 37 n Staphyll cus - 73

■ Entercbacteriaceae- 58

■ Gama-p roteo bacteria 42

■ Bacterioidetes- 28

■ alpha-proteobacteria - 15

■ Delta-proteobacteria - 8

□ Epsiion-prciteobacteria- 2

Figure 3.1. Distribution of best BLASTp hits of L. monocytogenes EGDe proteins among bacterial genera or higher-order taxonomic groups. (A color version of this figure appears between pages 196 and 197.)

remaining genes among the group of low GC Gram positive bacteria, the number of hits to proteins of each of the genera represented in this group depends on both phylogenetic distance and genome size. Accordingly, the highest number of best hits of Listeria proteins after those having their best hits with bacilli proteins is found with enterococci proteins, and the third with clostridia proteins. Clostridia are third although streptococci are phylogenetically more closely related, because of their larger genome size as compared to streptococci. This could be due to gene loss in particular in the flexible gene pool of streptococci related to the adaptation to the mammalian host environment. Surprisingly only 19 genes have a best BLASTp hit with proteins belonging to the group of Grampositive Actinobacteria. Among the bacterial species more distantly related to Listeria than the above mentioned, a high proportion of best hits (58 genes) was observed with proteins similar to proteins encoded by Enterobacteriaceae. These genes or some of them might have been acquired from Enterobacteri-aceae by horizontal gene transfer. For example, among these 58 genes, 24 are part of the vitamin B12 biosynthesis, propanediol, and ethanolamine utilization gene cluster, which have already been suggested to be horizontally transferred between these two groups of bacteria (Buchrieser et al. 2003). These genes are involved in anaerobic utilization of different carbon sources, which may contribute to the fitness of Listeria in the gut, like for some of the Enterobac-teriaceae. They also could be important for growth in vegetable waste such as silage known to be favorable for Listeria growth. Further examples for putative horizontal transfer from Enterobacteriaceae are three operons involved in sugar

Figure 3.1. Distribution of best BLASTp hits of L. monocytogenes EGDe proteins among bacterial genera or higher-order taxonomic groups. (A color version of this figure appears between pages 196 and 197.)

metabolism. The first operon, lmo2133-2137, is predicted to be implicated in fructose metabolism as it encodes two type II fructose 1-6 biphospahe aldolases. One is a Bacillus type and the second one is highly similar to the E. coli one. The second cluster, lmo2761-2764, encodes a beta-glucoside utilization operon and the third, lmo2834-2838, encodes four genes, probably also involved in sugar import and catabolism. This observation is in agreement with a shared growth environment for these two groups of strains, which is favorable for genetic exchange between strains.

It has been proposed that, in the environment, as for Legionella, unicellular eukaryotic organisms, like amoeba, algae, or protozoa, may be hosts for L. monocytogenes growth and survival (Gray et al. 2006). In the case of Legionella pneumophila, genome analysis revealed the abundance of genes encoding eukaryotic like proteins best explained by gene acquisition from the eukaryotic hosts (Cazalet et al. 2004). In the case of L. monocytogenes, we did not identify a single protein with a clear eukaryotic origin (i.e., a best BLASTp with an eukaryotic sequence) clearly indicating that L. monocytogenes does not interact so closely and for such a long time with a eukaryotic host as does Legionella species. In agreement with this observation, we observed a single gene (lmo1635) encoding a protein with a best BLASTp hit with an L. pneumophila protein. Overall, 91% of the Listeria genes other than phage genes have a best-BLASTp hit with proteins of low GC Gram positive bacteria. This strongly indicates that horizontal gene transfer to L. monocytogenes takes place mainly among closely related species.

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