Biofilms of the phylloplane

Question 1

the below ground habitate colonised by microbes is called the….

Answer 1

rhizosphere

Question 2

the aerial habitate colonized by microbed is called the…

Answer 2

phyllosphere

Question 3

what is the phylloplane?

Answer 3

the leaf surface
(where microbes live)

Question 4

what are the inhabitants of the phylloplane called?

Answer 4

epiphytes

Question 5

what is the approximate terrestrial leaf surface area?

Answer 5

~640 million sq km

Question 6

what hostile fluctuating environmnetal stresses do phylloplanes face?

Answer 6

UV, temp, dessication

Question 7

what are the two most abundant species on leaves of perennial rye (Lolium perenne)?

Answer 7

Pseudomonas fluorescens 20.12%
Xanthomonas campestris 19.64%

Question 8

what are the most abundant species on leaves of olive (Olea europea)

Answer 8

Pseudomonas syringae 51.0%
Xanthomonas campestris 6.7%

Question 9

what fresh produce is E.coli O157:H7 found on?

Answer 9

apple juice, bean sprouts, cabbage, celery, coriander, cress, lettuce

Question 10

what fresh produce is salmonella found on ?

Answer 10

Aubergine, bean sprouts, celery, cabbage, cauliflower, fennel, lettuce, orange juice, spinach, spring onions, strawberries, tomato, watermelon

Question 11

what fresh produce is Campylobacter spp found on?

Answer 11

Lettuce, mushrooms, potatoes, parsley, pepper, spinach, spring onions, watercress

Question 12

what fresh produce is Listeria monocytogenes found on?

Answer 12

Aubergine, bean sprouts, cabbage, cucumber, lettuce, mushrooms, potatoes, radishes, tomato

Question 13

what fresh produce is shigella found on?

Answer 13

Celery, lettuce, melon, parsley

Question 14

give some examples of pathogen vehicles onto the leaf surface

Answer 14

• compost
• irrigation water
• feral pig/pigeons

Question 15

what are the disadvantages to ex situ sample preparation and culture recovery methods

Answer 15

• Non-culturable species and strains exist
• Sub-lethally stressed cells may not grow within a given incubation period, or indeed at all.
• Accuracy depends on efficient recovery from the sample being tested – SAMPLE PREPARATION IS VERY IMPORTANT – the Pulsifier

Question 16

what microscope is used for in situ detection?

Answer 16

EDICEF microscopy

Question 17

what leaf structures can be seen in EDIC microscopy?

Answer 17

epidermal cells
stroma
leaf vein cells

Question 18

what is the stroma?

Answer 18

air pore for diffusion of gases in and out of leaf

Question 19

what the function of leaf vein cells?

Answer 19

transport of water and nutrients

Question 20

why does chlorine end up in salad washing processes?

Answer 20

Salad production occurs on the east side of the UK, which is sunny and dry and thus the production process requires lots of water. to minimise water usage the companies recycle the water and add chlorine to it to keep it clean

Question 21

what are GFP bacteria

how are they useful?

Answer 21

strains of bacteria engineered to produce Green Fluorescent Protein

• This makes each bacterial cell fluoresce bright green when viewed using fluorescence microscopy
• These bacteria can be tracked and counted wherever they colonise, and are a very useful tool across microbiology

Question 22

describe the localisation of bacteria on leaves after innoculation

Answer 22

• Immediately after inoculation: many bacteria in cell margins but also well spread over leaf surface
• Within 2 hours: bacteria predominantly present at cell margins and within stomatal apertures

Question 23

give some potential attachments mechanisms of bacteria to the phylloplane

Answer 23

Flagellum
Pilli
Hydrogen bonding
Ionic bonding
Cellulose

Question 24

how can you tell the difference between pilli and fimbriae

Answer 24

Pilli tend to be the longer ones in fewer numbers and the fimbrae are shorter and more numerous

Question 25

give example of salmonella attachment mutants

Answer 25

* Δcrl and ΔcsgB (curli (fimbrei) specific gene) mutant strains partly lacking curli fimbriae * ΔrpoS and ΔcrlΔcsgB mutant strains lacking curli fimbriae * ΔflhC mutant strains lacking flagella

Question 26

what is the difference in attachment of curli fimbriae mutants to abiotic and biotic surfaces

Answer 26

When you do these experiments with plastic wells (abiotic) then the knockout mutants do their jobs, affenting attachment and biofilm formation, however when you do it on real world surface (leaf) then the mutants don’t affect growth that significantly

Question 27

which salmonella mutant showed reduced attachment to the phylloplane?

Answer 27

flhC mutant (lacking flagella)

Question 28

what important genetic regions were identified through ID attachment defective transposon mutants?

Answer 28

* intergenic region between agfB (also called csgB) - the surface-exposed aggregative fimbria (curli) nucleator, * agfD (csgD), a transcriptional regulator of the LuxR superfamily (quorum sensing), * and rpoS, the stationary-phase sigma factor. ## Footnote Both AgfD and RpoS have been reported to regulate curli and cellulose production and RpoS regulates other adhesins such as pili.

Question 29

how do mutations of the agfA curli subunit and agfB differentially affect attachment to plant tissues?

Answer 29

The agfA (csgA) mutation was not reduced in ability to attach to or colonize alfalfa sprouts, whereas the agfB (csgB) mutation was reduced. * Thus, agfB (csgB) alone can play a role in attachment of S. enterica to plant tissue: thin aggregative fimbriae

Question 30

what is BapA and how does it affect attachment?

Answer 30

BapA: Biofilm associated protein interacts with calcium to facilitate binding

Question 31

what is the functional consequences of csgD interaction with adrA

Answer 31

intiates cellulose formation

Question 32

how does RpoS controll production of cellulose and cyclic di gmp

Answer 32

rpos controls the expression of cgsd which activates expression of adrA AdrA is part of control of cellulose production and controls cylic di gmp

Question 33

what are the benefits to bacteria seeking refuge in cell margins and stoma?

Answer 33

* These refuges are less hostile in terms of water availability and protection from solar irradiation, sanitisers and washing/detachment methods