Essential bacterial genes and their use

Details Essential bacterial genes and their use Essential bacterial genes and their use

1998-12-30

2002-08-20

Smith, Lynette R. F (Department: 1645)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S006120, C435S233000, C435S034000, C435S069100, C536S023200, C514S04400A

Reexamination Certificate

active

06437108

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to essential bacterial genes and their use in identifying antibacterial agents.
Bacterial infections may be cutaneous, subcutaneous, or systemic. Opportunistic bacterial infections proliferate, especially in patients afflicted with AIDS or other diseases that compromise the immune system. The bacterium
Streptococcus pneumonia
typically infects the respiratory tract and can cause lobar pneumonia, as well as meningitis, sinusitis, and other infections.
SUMMARY OF THE INVENTION
The invention is based on the discovery of 23 genes in the bacterium
Streptococcus pneumoniae
, and a related gene in the bacterium
Bacillus subtilis
, that are located within operons that are essential for survival. These 23 Streptococcus genes are referred to herein as “GEP genes” (which stands for general essential protein); for convenience, the polypeptides encoded by these genes are referred to herein as “GEP polypeptides.” Each GEP gene is located within an operon that contains a gene that is essential for survival of
Streptococcus pneumoniae
; the essential gene can be the GEP gene or another gene located within the same operon. Bacterial operons contain several genes that are related, e.g., with respect to function or biochemical pathway. Transcription of an operon leads to the production of a single transcript in which multiple coding regions are linked. Thus, an operon containing one or more essential genes can be considered an “essential operon,” since disruption of expression of one gene located within the operon will interfere with expression of the other genes in the operon. Each coding region of the transcript is separately translated into an individual polypeptide by ribosomes that initiate translation at multiple points along the transcript. Having identified one gene in the operon, one can readily identify and sequence the other genes located within the operon.
The genes encoding the GEP polypeptides are useful molecular tools for identifying similar genes in pathogenic microorganisms, such as pathogenic strains of Bacillus. In addition, the operons containing genes encoding GEP polypeptides, and the polypeptides encoded by such operons, are useful targets for identifying compounds that are inhibitors of the pathogens in which the GEP polypeptides are expressed. Such inhibitors inhibit bacterial growth by being bacteriostatic (e.g., inhibiting reproduction or cell division) or by being bacteriocidal (i.e., by causing cell death).
The invention, therefore, features an isolated polypeptide encoded by a nucleic acid located within an operon encoding a GEP polypeptide, termed gep103, having the amino acid sequence set forth in SEQ ID NO:1, or conservative variations thereof. An isolated operon comprising a nucleic acid encoding gep103 also is included within the invention. In addition, the invention includes an isolated nucleic acid of (a) an operon comprising the sequence of SEQ ID NO:2, as depicted in
FIG. 1
, or degenerate variants thereof; (b) an operon comprising the sequence of SEQ ID NO:2, or degenerate variants thereof, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) that are at least 15 base pairs in length and that hybridize under stringent conditions to genomic DNA encoding the polypeptide of SEQ ID NO:1. As described above for gep103, other nucleic acids and polypeptides encoded by nucleic acids located within operons encoding GEP polypeptides are included within the invention, including: (a) operons comprising the nucleic acids represented by the SEQ ID NOs. listed below, as depicted in the Figures listed below, or degenerate variants thereof; (b) operons comprising the nucleic acids represented by the SEQ ID NOs. listed below, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) that are at least 15 base pairs in length and that hybridize under stringent conditions to genomic DNA 5 encoding the polypeptides represented by the SEQ ID NOs. listed below.
TABLE 1
GEP nucleic acids and polypeptides
SEQ ID
SEQ ID
NO. OF
NO. OF
THE NON-
THE CODING
CODING
GEP
STRAND
STRAND
NUCLEIC
SEQ ID
OF THE
OF THE
ACID OR
NO. OF
NUCLEIC
NUCLEIC
POLY-
FIG.
AMINO ACID
ACID
ACID
PEPTIDE
NO.
SEQUENCE
SEQUENCE
SEQUENCE
gep103
1
1
2
3
gep1119
2A-B
4
5
6
gep1122
3A-D
7
8
9
gep1315
4A-B
10
11
12
gep1493
5
13
14
15
gep1507
6
16
17
18
gep1511
7A-B
19
20
21
gep1518
8A-C
22
23
24
gep1546
9
25
26
27
gep1551
10A-B
28
29
30
gep1561
11A-B
31
32
33
gep1580
12A-B
34
35
36
gep1713
13A-B
37
38
39
gep222
14A-B
40
41
42
gep2283
15A-B
43
44
45
gep273
16A-B
46
47
48
gep286
17A-B
49
50
51
gep311
18A-B
52
53
54
gep3262
19
55
56
57
gep3387
20
58
59
60
gep47
21A-C
61
62
63
gep61
22A-B
64
65
66
gep76
23A-B
67
68
69
The invention also includes allelic variants (i.e., genes encoding isozymes) of the genes located within operons encoding the GEP polypeptides listed above. For example, the invention includes a gene that encodes a GEP polypeptide but which gene includes one or more point mutations, deletions, promotor variants, or splice site variants, provided that the resulting GEP polypeptide functions as a GEP polypeptide (e.g., as determined in a conventional complementation assay).
Identification of these GEP genes and the determination that they are located within operons containing an essential gene allows homologs of the GEP genes to be found in other organisms strains of Streptococcus. Also, orthologs of these genes can be identified in other species (e.g., Bacillus sp.). While “homologs” are structurally similar genes contained within a species, “orthologs” are functionally equivalent genes from other species (within or outside of a given genus, e.g., from
Bacillus subtilis
or
E. coli
). Such homologs and orthologs are expected to be located within operons that are essential for survival. Such homologous and orthologous genes and polypeptides can be used to identify compounds that inhibit the growth of the host organism (e.g., compounds that are bacteriocidal or bacteriostatic against pathogenic strains of the organism). Homologous and orthologous genes and polypeptides that are essential for survival can serve as targets for identifying a broad spectrum of antibacterial agents.
An ortholog of gep1493, termed B-yneS, has been identified in
B. subtilis
and is essential for survival of
B. subtilis
. The amino acid sequence (SEQ ID NO: 70), coding sequence (SEQ ID NO:71), and non-coding sequence (SEQ ID NO:72) of B-yneS is set forth in
FIGS. 24A-B
. As with the other polypeptides and genes disclosed herein, the B-yneS polypeptide and gene can be used in the methods described herein to identify antibacterial agents.
The term gep103 polypeptide or gene as used herein is intended to include the polypeptide and gene set forth in
FIG. 1
herein, as well as homologs of the sequences set forth in FIG.
1
. Also encompassed by the term gep103 gene are degenerate variants of the nucleic acid sequence set forth in
FIG. 1
(SEQ ID NO:2). Degenerate variants of a nucleic acid sequence exist because of the degeneracy of the amino acid code; thus, those sequences that vary from the sequence represented by SEQ ID NO:2, but which nonetheless encode a gep103 polypeptide are included within the invention. Likewise, because of the similarity in the structures of amino acids, conservative variations (as described herein) can be made in the amino acid sequence of the gep103 polypeptide while retaining the function of the polypeptide (e.g., as determined in a conventional complementation assay). Other gep103 polypeptides and genes identified in additional Streptococcus strains may be such conservative variations or degenerate variants of the particular gep103 polypeptide and nucleic acid set forth in
FIG. 1
(SEQ ID NOs:1 and 2, respectively). The gep103 polypeptide and gene share at least 80%, e.g., 90%, sequence identity with SEQ ID NOs:1 and 2, respectively. Regardless of the percent sequence identity between the gep103 sequence and the sequence repre

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