Wednesday, April 26, 2006

Study Questions relating to mechanisms of nutrient uptake by bacteria.

Q1. How are proton gradients created?

Q2. What is the role performed by outer-membrane pores (porins) of gram-negative bacteria?

Q3. Give two examples of facilitated diffusion as a substrate uptake mechanism in bacteria?

Q4. How is facilitated diffusion selective?

Q5. The group translocation process accomplishes transport by chemically modifying the solute, which arrives inside the cell as a different molecule.

Give an example of substrate uptake by group translocation in bacteria, and describe the source of energy used for active uptake.

Your responses in comments please.(They can be anonymous!)

Sunday, April 23, 2006

Ammonia assimilation is an important part of the biosynthesis stage of growth metabolism.

After the fueling step of growth metabolism has occurred, ammonia is incorporated into metabolites. This part of biosynthesis is known as nitrogen assimilation.

Study question:

How much of the cell is nitrogen?

Two alternative assimilation routes for nitrogen:

Although some nutritional sources of nitrogen for bacterial cells (e.g. nitrate) are in a more oxidised chemical state than ammonia, these are uniformly converted to ammonia before assimilation, and almost all cell nitrogen is at the same oxidation state as ammonia. Ammonia is fully reduced, fitting in with the concept that early life evolved in an oxygen free environment.

Escherichia coli
has two different first steps for incorporating ammonia into building blocks for polymers such as proteins and nucleic acids, each alternative relying on a different enzyme.

One of these alternative uses glutamate as an acceptor of ammonia, ATP as a co-substrate, and the enzyme glutamine synthetase to catalise the reaction:

The other alternative first step for ammonia assimilation uses the metobolite 2-oxoglutarate (also called alpha ketoglurate), to accept ammonia, NADPH and the enzyme GDH.

Study Questions:

What are the products of the two alternative first steps in ammonia assimilation?

How do the two reactions differ in terms of reaction products?

More importantly, what are the different advantages and opportunities offered by the two alternatives to the cell in its survival under varous different environmental challenges?

Reference Sources:

Chapter 7, Biosynthesis, in Microbe, Schaechter 2006

EcoCyc, Encyclopedia of Escherichia coli K-12 Genes and Metabolism

Why does Escherichia coli have two primary pathways for synthesis of glutamate?
Helling RB.
J Bacteriol. 1994 Aug;176(15):4664-8. Erratum J Bacteriol 1997 Jul;179(13):4455.

Saturday, April 22, 2006

Biosynthesis Stage of Growth Metabolism.

Biosynthesis of building blocks for polymers - such as amino acids and nucleotides- follows the Fueling reaction stage of microbial growth metabolism.

Biosynthesis differs from the Fueling stage in that nitrogen (as ammonia) and sometimes sulphur are assimilated into the molecules of metabolism. Nitrogen is not found in the fuelling precursor metabolites.

Generally the end products of biosynthesis are more reduced than the precursor metabolites, and this reduction is achieved by use of NADPH to donate hydrogen. Additionally ATP is also used, so that ATP and NADPH provide the driving force for biosynthesis.

Quoting from Schaechter 2006 Chapter 7:

Biosynthesis, then, more than fueling, reflects the essentially close relation of microbes to plants and animals. While the fueling metabolism of microbes expresses the potential for life forms to scavenge for food and thereby recycle organic material, the biosynthetic metabolism of microbes reminds us of what we have lost in evolving into beings higher in the food chain.

Study Question:
Explain in what way the biosynthesis of amino acids and other nitrogen-containing components of human cells depends ultimately on the biochemical activities of Earth's prokaryotes.

Responses in comments please.

Tuesday, April 11, 2006

The best way to find information about bacteria is through effective use of both publication and gene databases.

La Cosa Nostra Genetica

This post is to help people use computer searches effectively to find biological information about cell functions.

When it comes to functional information on singled celled organisms that have a genome sequence completed, a very powerful way forward is to take these steps
  1. Start by using a relevant words searche via PubMed or equivalent service, to locate an appropriate protein sequence in a sequence data base (say at NCBI website),and
  2. Then use the retrieved protein sequence to BLASTP for relevant other genes in any organism of interest.
  3. Search the BLASTP output for clues to relevance of for new ideas.
The reason why this approach is powerful is that it enables natural evolution to be traced in silico. Evolutionary traces make definite connections between unknown functions in one putative gene to known functions in other well studied genes, and are supported by huge and constantly growing genome databases.

To illustrate this, Microbe Pundit will show the trail of results obtained in a student discussion about secretion of biologically active compounds by Streptomyces avermitilis.

Pundit started by assuming Quorum sensing systems would be involved in secretion of important compounds in this organism- the question is how to find the genes involved.

First find a studied secretion system in any Streptomycete. (Evolutionary relatedness neans that all Streptomycete species will share many important genes).

To find this, go to Pubmed, the guide for published medical scientific papers, and type in "QUORUM SENSING STREPTOMYCES" as a search item, press buttons and scan the output for a better more specific lead in the form of a relevant paper.

This is computer detective work in action. It helps if you imagine you are Sherlock Holmes or Mrs Marples.

The trick is the use you own brain to find useful further clues in this computer output.

This approach lead me to the following paper:

1: J Bacteriol. 2005 Jan;187(1):135-42.

Dual transcriptional control of amfTSBA, which regulates the onset of cellular differentiation in Streptomyces griseus.
Ueda K, Takano H, Nishimoto M, Inaba H, Beppu T.

The amf gene cluster encodes a probable secretion system for a peptidic morphogen, AmfS, which induces aerial mycelium formation in Streptomyces griseus. Here we examined the transcriptional control mechanism for the promoter preceding amfT (PamfT) directing the transcription of the amfTSBA operon.
High-resolution S1 analysis mapped a transcriptional start point at 31 nucleotides upstream of the translational start codon of amfT. Low-resolution analysis showed that PamfT is developmentally regulated in the wild type and completely abolished in an amfR mutant. The -35 region of PamfT contained the consensus sequence for the binding of BldD, a pleiotropic negative regulator for morphological and physiological development in Streptomyces coelicolor A3(2).
The cloned bldD locus of S. griseus showed high sequence similarity to the S. coelicolor counterpart. Transcription of bldD occurred constitutively in both the wild type and an A-factor-deficient mutant of S. griseus, which suggests that the regulatory role of BldD is independent of A-factor. The gel retardation
assay revealed that purified BldD and AmfR recombinant proteins specifically bind PamfT. Overproduction of BldD in the wild-type cell conferred a bald phenotype (defective in aerial growth and streptomycin production) and caused marked repression of PamfT activity. An amfT-depleted mutant also showed a bald
phenotype but PamfT activity was not affected. Both the bldD-overproducing wild-type strain and the amfT mutant were unable to induce aerial growth of an amfS mutant in a cross-feeding assay, which indicates that these strains are defective in the production of an active AmfS peptide. The results overall suggests that two independent regulators, AmfR and BldD, control PamfT activity via direct binding to determine the transcriptional level of the amf operon responsible for
the production and secretion of AmfS peptide, which induces the erection of aerial hyphae in S. griseus.

The bolded sections are key facts (always skip over details at this stage you are trolling for pearls).

Pundit assumed one of these amf genes must code for a transport (peptide secretion) system. Lets assume it is gene amfB

Next go to a protein sequence database that takes word query searches such as this one at NCBI.

Type in amfB and press the right buttons, use common sense and you will find this entry:

LOCUS NP_828677 595 aa linear BCT 16-FEB-2006
ABC transporter ATP-binding membrane translocator, AmfB [Streptomyces avermitilis MA-4680].
VERSION NP_828677.1 GI:29834043

SOURCE Streptomyces avermitilis MA-4680
ORGANISM Streptomyces avermitilis MA-4680
Bacteria; Actinobacteria; Actinobacteridae; Actinomycetales;
Streptomycineae; Streptomycetaceae; Streptomyces.
AUTHORS Ikeda,H., Ishikawa,J., Hanamoto,A., Shinose,M., Kikuchi,H.,
Shiba,T., Sakaki,Y., Hattori,M. and Omura,S.
TITLE Complete genome sequence and comparative analysis of the industrial
microorganism Streptomyces avermitilis
JOURNAL Nat. Biotechnol. 21 (5), 526-531 (2003)
PUBMED 12692562
AUTHORS Omura,S., Ikeda,H., Ishikawa,J., Hanamoto,A., Takahashi,C.,
Shinose,M., Takahashi,Y., Horikawa,H., Nakazawa,H., Osonoe,T.,
Kikuchi,H., Shiba,T., Sakaki,Y. and Hattori,M.
TITLE Genome sequence of an industrial microorganism Streptomyces
avermitilis: deducing the ability of producing secondary
JOURNAL Proc. Natl. Acad. Sci. U.S.A. 98 (21), 12215-12220 (2001)
PUBMED 11572948


FEATURES Location/Qualifiers
source 1..595
/organism="Streptomyces avermitilis MA-4680"
/strain="MA-4680; ATCC 31267; NCIMB 12804; NRRL 8165"
Protein 1..595
/product="ABC transporter ATP-binding membrane
translocator, AmfB"
Region 82..589
/region_name="ABC-type multidrug transport system, ATPase
and permease components [Defense mechanisms]"
Region 375..570
/region_name="ABC (ATP-binding cassette) transporter
nucleotide-binding domain"
CDS 1..595
/note="AmfB/RamA homolog protein"


1 mrghsrmktp sghgaapdgt gaadeaaart llrsaarhsr srcvalcltt aaasgaslll
61 paalgraldl lltrpgdaag thwvlwctgl vllialldac htvlagttda ratawlrqrl
121 vghvlavgpr agerfgpgel varlvgnaaq agtapataat llaalagpvg avvalglidp
181 llaavflgga pvltlllraf ardssqcvar yqdvqgriag alaeaiggar tiaaggtadk
241 evarilrplp elsregrrmw rvqgraaaqa vavapllqlg vvavggvllv hhrlsvgell
301 aasryavlat gvgvlvgqls gliraraaar rlgevltepa pvygtrqlpp gegrlelrsv
361 tvrrggrtvl dgvdlvvpag rtvavvgrsg sgksllaala grladpddgh vlldgvplrd
421 ldrtalrrav ghaferpall gdtiedtiaf gipspppdrv rqaaatarad sfvrrlpdgy
481 atpcaeapls ggecqrlgla rafahdsrll vlddalssld tvterhitea llrhtpgssr
541 liiahrvsta aradavvwla agrvravgth aelwrsaayr evfgssgter nggag

This means somebody has already annotated a similar putative gene to amfB in S. avermititis. The implied membrane located active transport sytem is of the ABC or ATP binding cassette type.

There are hundreds of known ABC-transporters. Some secrete compounds, other import compounds.

The next question is to ask what are the evolutionarily related transport systems to this one?, particularly one that have been investigated in the lab. (Hypothetical functions predicted by computers are "dime-a-hundred". Experimentally characterised functions are pearls.)

For this you take the protein sequence (bolded above) and go to the BLASTP search tool here.

Paste in the protein sequence from the entry above and again push buttons.

The output is huge.

This a a small selection of what you get:
gi|29834043|ref|NP_828677.1|  ABC transporter ATP-binding memb...   686    0.0    Gene info
gi|4928927|gb|AAD33775.1|  putative ATP binding membrane trans...   275    4e-72
gi|21224979|ref|NP_630758.1|  ABC transporter ATP-binding prot...   273    2e-71  Gene info
gi|432992|gb|AAA21388.1|  potential ATP-binding membrane transpor   273    2e-71
gi|31044106|dbj|BAA33538.2|  membrane translocator [Streptomyces    267    1e-69
gi|50905835|ref|XP_464406.1|  putative multidrug resistance p-...   117    2e-24  Gene info
gi|34913530|ref|NP_918112.1|  putative multidrug resistance pr...   115    6e-24  Gene info
gi|85813525|emb|CAF33031.1|  putative ABC-type aminoglycoside ...   114    2e-23
gi|35214709|dbj|BAC92076.1|  HlyB/MsbA family ABC transporter ...   108    5e-22  Gene info
gi|7023646|dbj|BAA92038.1|  unnamed protein product [Homo sapiens   108    9e-22  Gene info
gi|27378906|ref|NP_770435.1|  ABC transporter HlyB/MsbA family...   106    3e-21  Gene info
gi|7688707|gb|AAF67494.1|  NovA [Streptomyces caeruleus]            102    5e-20
gi|26991605|ref|NP_747030.1|  toxin secretion ABC transporter ...   102    5e-20  Gene info
gi|2633146|emb|CAB12651.1|  yfiC [Bacillus subtilis subsp. sub...   102    5e-20  Gene info
gi|85813784|emb|CAF31837.1|  putative hygromycin B exporter [S...  99.4    4e-19
Try pressing a few of the hyperlinks above to inspect database entry details.
The hits that are connected with export of antibiotics in Streptomyces are the most interesting finds- a reward for due diligence.

Sherlock Holmes and the Pundit can find these easily in the output.

Computers by themselves are stupid and can't do this well.
Humans are also needed to hop effectively between databases, as shown above.
After a while you will get good at this thing of ours, so lets call it La Cosa Nostra Genetica

Il Signor Pundit