Genes Associated with Biofilm

Formation in Mycobacterium
smegmatis

Molly D. McNab
Oregon State University
College of Veterinary Medicine
Department of Biomedical Sciences
Summer 2003
Mentor: Dr. Luiz Bermudez
What is Biofilm?
 Biofilms are multicellular aggregates of
bacteria and yeast that congregate on
surfaces.
 Biofilm may form on any surface exposed
to biofilm-forming bacteria and some
amount of water.
 Biofilms are formed to protect the
bacteria from host defenses, antibiotics,
and from harsh environmental conditions.
 Where are Biofilms Found?
Biofilms are found almost
everywhere in nature,
including rivers, lakes,
soil, water pipes, and even
inside the human body.

A common type of bacterial

biofilm-responsible for plaque.

Bacterial biofilms are often a cause of

infections associated with medical
implants such as catheters and IV lines
and other medical devices.
Why Research Biofilms?
 Due to the morphology of biofilms,
bacteria capable of forming them are
highly resistant to antibiotics, making
treatment very difficult.
 In the US alone, one million nosocomial
(hospital acquired) infections each year
are caused by bacterial biofilms, leading
to longer hospitalization, surgery, and
even death.
Biofilms and Infections:
 Biofilms are responsible for Otitis Media, the
most common acute ear infection.
 Biofilms play a role in Bacterial Endocarditis
(infection of the inner surface of the heart and its
valves).
 Biofilms form frequently in patients with Cystic
Fibrosis (a chronic disorder resulting in increased
susceptibility to serious lung infections).
 Biofilms also play a role in Legionnaire's
disease (an acute respiratory infection resulting
from the aspiration of clumps of Legionnella
biofilms detached from air and water
heating/cooling and distribution systems).
How are Biofilms Formed?

Biofilm formation relies on an exchange of

chemical signals between cells in a process
known as “quorum sensing.”
Quorum Sensing
 Quorum sensing is required for
natural biofilm formation.
 When enough bacteria (a quorum)
are present, diffusible signal
molecules produced by the bacteria
allow for communication with others
in order to coordinate their
behavior.
Mycobacteria: An Overview
 There are >70 species of mycobacteria,
many of which are human pathogens.
 Of these, three are major pathogens:
 Mycobacterium tuberculosis
 Mycobacterium leprae
 Mycobacterium avium
 Many species of mycobacteria are found
in the environment where biofilm
formation is demonstrated.
Incidence of Mycobacterial Infections
 8-12 million new infections of M. tuberculosis are
reported per year, particularly in developing
countries.
 2-3 million people die from TB each year
 Antibiotic resistant strains of M. tuberculosis are
very common and cause great public health
concern.
 In the USA, environmental mycobacteria are more
common than M. tuberculosis in a clinical setting;
this is due to their association with AIDS and the
low incidence of TB in this country.
 M. avium is an environmental mycobacteria that is
capable of forming biofilm and often infects AIDS
patients and those with chronic pulmonary
diseases.
Mycobacterium smegmatis
 Avirulent mycobacterium
 Ability to form biofilm
 Found in the environment
 Shares many properties with other
more virulent mycobacteria
 Grows easily in a laboratory setting
and is fairly receptive to genetic
manipulation
My Research

 Using Mycobacterium smegmatis as a

model, I will investigate the genes that
are important for biofilm formation in
other more virulent mycobacteria.
 Design of My Plasmid:

M. avium Library Promoter-less GFP

pEMC 1

Kanamycin Resistance
Mycobacterium Origin of Replication
Step One:
 In a 96-well plate (shown below) grow
bacteria (5 colonies per well), transferred
directly from an already prepared GFP
promoter library, in 200µL 7H9 growth
media with Kanamycin (50µg/mL).
Incubate at 37°C for 3-4 days to increase
bacterial concentration.
 Step Two:
 After 3-4 days, transfer 100µL from each
well into a 96-well Polyvinyl Chloride (PVC)
plate, which promotes biofilm formation.
 Store the PVC plate at room temperature
with slight agitation.
 Read the intensity of GFP expression of
each well on day one, to use as a control,
and each following day up to day five.
Step Three:
 Analyze the results of the GFP
expression assay by comparing day
five with the day one controls.
 Individual wells whose GFP
expression increases by at least two
times are isolated from the original
96-well plates, then plated on 7H11
agar (with OADC and Km 50) and
allowed sufficient time for growth.
 Sample GFP Expression Assay Results

117 127 115 122 113 141 123 115 103 131

53 63 56 61 65 68 65 66 67 75

102 101 63 67 74 94 84 82 83 105

74 58 66 67 69 68 62 65 66 117

88 68 116 69 80 116 72 73 68 75
Day One (Control)
56 55 56 59 56 58 60 60 63 76

61 65 79 58 86 103 60 90 122 139

73 84 63 66 67 63 62 82 67 71

141 133 116 142 121 141 154 152 141 143

64 69 51 65 77 65 59 59 62 68

119 114 69 94 88 117 158 89 133 130

101 68 71 91 92 76 66 57 61 123
Day Five
101 98 157 96 93 122 95 79 80 87

59 49 50 52 51 55 53 53 58 84

66 60 119 51 109 124 57 90 144 149

88 107 62 66 80 60 58 89 67 98
 Analysis of Results

1.44 1.55 1.21 1.05 1.01 1.16 1.07 1.00 1.25 1.32 1.37 1.09

1.17 1.60 1.21 1.10 0.91 1.07 1.18 0.96 0.91 0.89 0.93 0.91

1.35 1.66 1.17 1.13 1.10 1.40 1.19 1.24 2.00 1.09 1.60 1.24

2.05 1.52 1.36 1.17 1.08 1.36 1.33 1.12 1.06 0.88 0.92 1.05

1.56 1.21 1.15 1.44 1.35 1.39 1.16 1.05 1.32 1.08 1.18 1.16

1.27 1.07 1.05 0.89 0.89 0.88 0.91 0.95 0.88 0.88 0.92 1.11

1.72 1.31 1.08 0.92 1.51 0.88 1.27 1.20 0.95 1.00 1.18 1.07

1.67 1.74 1.21 1.27 0.98 1.00 1.19 0.95 0.94 1.09 1.00 1.38
Step Four:
 32 individual colonies from
each well are picked up and
transferred to a 96-well plate
with the 7H9 + Km50 + 10%
OADC growth medium
described in step one.
 This is then incubated at
37°C for 3-4 days.
 Steps one and two are
repeated with the new plate
(1 colony per well)
Step Five:
 Analyze the results of the GFP
expression assay by comparing day
five with the day one controls.
 Individual wells whose GFP
expression is increased by at least
two times are isolated again and
prepared for plasmid extraction.
Step Six:
 The plasmids from the wells with
the most green intensity are
extracted, transformed into E. coli,
extracted again, and then sent for
sequencing.
 The sequences are then matched up
with the genomes M. avium and M.
tuberculosis to find the specific gene
or protein and its function.
Results:
Genes found to play a role in biofilm
formation:
 Glycosyltransferase (CDC 1551)
 GuaB2 (H37Rv) IMPDH (CDC 1551)
 Rv0538, Rv0539 (H37Rv)
 Rv 3412, Rv 3413c (H37Rv)
 Rv 3526 (H37Rv)
 Rv 0359, Rv0358 (H37Rv)
Discussion:
 The absence of glycopeptidolipids (GPLs) in
the outermost layer of the cell wall
abolishes the ability of M. smegmatis and
M. avium to form biofilm on PVC.1
 Glycosyltransferase (CDC 1551) catalyzes
the addition of Rha (3-O-Me-rhamnose) to
6-d-Tal (6-deoxytalose) and is essential for
the expression of mature GPLs.2
1Recht, J. et al. Glycopeptidolipid Acetlylation Affects Sliding Motility and Biofilm formation in Mycobacterium Smegmatis. J. Bacteriology.
Oct 2001. p. 5718-5724.
2 Torsten M. et al. Identification and Recombinant Expression of a Mycobacterium avium Rhamnosyltransferase Gene (rtfA) Involved in

Glycopeptidolipid Biosynthesis. J. Bacteriology. Nov 1998 p. 5567-5573

 Model of Mycobacterial Cell Wall

Lipoarabinomannon (LAM)
Glycopeptidolipid (GPL)

Trehalose
cell wall
Mycolic acid
cytoplasmic Peptidoglycan
membrane

cytoplasma

* GPL is associated with biofilm formation in mycobacteria

Discussion, continued:
 GuaB2 (H37Rv), which is the same as
IMPDH (CDC1551), is an inosine 5’
monophosphate dehydrogenase. This
protein catalyzes the first reaction unique
to GMP biosynthesis, which in turn
synthesizes GDP and is necessary for GPL
expression.
 IMPDH also has a key role in the growth
of many cell types.
Escobar-Henriques, M. et al. Transcriptional Regulation of the Yeast GMP Synthesis Pathway by Its End Products. J of Biological
Chemistry. 276:2 pp. 1523-1530. 2001.
Discussion, continued:
 Rv0539 (H37Rv) is a probable glycosyltransferase
and, as mentioned before, is important for the
expression of mature GPLs.
 Rv 3413c (H37Rv) is a probable Alanine and
Proline rich protein, function unknown
 Rv 3526 (H37Rv) is a possible oxidoreductase and
is probably involved in cellular metabolism.
 Rv 0359 (H37Rv) is a probable conserved integral
membrane protein
 Rv0358, Rv 3412, and Rv0538 (H37Rv) are
conserved hypothetical proteins, functions are not
yet known.
Conclusions:
 Several genes isolated through
research seem to play a role in
glycopeptidolipid (GPL) biosynthesis
or expression.
 The relationship of GPLs to
successful biofilm formation in M.
Avium and M. Smegmatis on PVC is
affirmed.
Acknowledgements
 HHMI
 Luiz Bermudez
 Yoshitaka Yamazaki
 OSU College of Veterinary Medicine